Hybridization promotion reagents

ABSTRACT

Novel reagents are provided along with methods for their use in nucleic acid hybridization assays for detecting target nucleic acids in samples. The most preferred reagent is a mixture of guanidinium thiocyanate and tetramethylammonium:Trifluoracetate and possesses many properties for facilitating hybridization between target nucleic acid and nucleic acid probes capable of binding thereto and surprisingly results in the phenomenon of superstoichiometric labeling.

This is a divisional of application Ser. No. 08/290,001, filed Aug. 11,1994, now U.S. Pat. No. 5,589,335, which is a continuation ofapplication Ser. No. 08/093,406, filed Jul. 19, 1993, now abandoned,which is a continuation of application Ser. No. 07/821,334, filed Jan.13, 1992, now abandoned, which is a continuation-in-part of applicationSer. No. 07/333,656 filed Apr. 5, 1989, now abandoned.

FIELD OF THE INVENTION

This invention pertains to the field of nucleic acid hybridizationassays and more specifically involves novel reagents for conducting andpromoting hybridization of nucleic acid probes with target nucleic acidsfrom a food or clinical sample.

BACKGROUND OF THE INVENTION

Specific association of complementary nucleic acid takes place in areaction commonly referred to as hybridization. This technique has madepossible the sensitive and specific detection, and the isolation, ofnucleic acid sequences. In this technique, one prepares a nucleic acidprobe which is complementary to a target nucleic acid, and then byselecting the appropriate conditions causes these two entities to bindto each other to form a hybrid. By judiciously choosing the reactionconditions, e.g. temperature, ionic strength and the like, one preventsnon-complementary nucleic acids from hybridizing. Hybrids can be formedon a solid support, in tissue sections or in solution. In the last casethey are subsequently separated from a mixture of nucleic acids by oneof a variety of methods and immobilized on a solid support. Theformation of hybrids can be followed if a "reporter" probe contains adetectable element, such as a radioactive atom, an enzyme withdetectable activity, a hapten to which an enzyme-conjugated antibody canbind, or generally any entity to which an enzyme-conjugated ligand canbind.

Since its discovery, nucleic acid hybridization has been usedextensively as a research tool. More recently, it has been put to use asa medical diagnostic technique, as a method for testing food for thepresence of pathogens, in agriculture, etc. In these fields,hybridization is in its infancy: there is an ongoing need to developimproved techniques, to make the assay methods more sensitive, morespecific, faster and easier to use. It is one aspect of the presentinvention to provide an improved reagent for use in hybridizationassays.

Most hybridization assays are done overnight with targets immobilized onfilters (Nygaard, A. P. and Hall, B. D. (1963) Biochem. Biophys. Res.Commun. 12, 98-104; Gillespie, D. and Spiegelman, S. (1965) J. Molec.Biol. 12, 829-842; Southern, E. M. (1975) J. Molec. Biol. 98, 503-517).Though these techniques have been extremely useful for research purposesand have made possible a variety of significant discoveries, a suitableclinical assay must be much more rapid (with results available within anhour or two of specimen collection), as well as being simpler (filterimmobilization is not trivial, especially with clinical samples) andmore sensitive.

The nucleic acids in a hybridization reaction generally have to behighly purified prior to immobilization, especially when non-radioactivedetection is used (Ruth, J. L. and Bryan, R. N. (1984) Fed. Proc. 43,2048; Kuritza, A. P., Getty, C. E., Shaughnessy, P., Hesse, R. andSalyers, A. A. (1986) J. Clin. Microbiol. 23, 343-349; Zwadyk, P.,Cooksey, R. C. and Thornsberry, C. (1986) Curr. Microbiol. 14, 95-100).While this is less of a major problem in a research setting, this isunacceptable in a clinical situation because nucleic acid purificationcan be a multi-step, time-consuming process, where an assay must berapid and simple.

It is another aspect of the present invention to provide a clinicalassay incorporating a rapid and simplified sample preparation step alongwith reagents suitable therefor. Gillespie (International ApplicationNo. WO 87/06621, dated Nov. 5, 1987) has described a method of carryingout nucleic acid hybridizations which does not require priorpurification and/or immobilization of solubilized target nucleic acids.In this method, guanidinium thiocyanate serves both to solubilize atarget nucleic acid and to permit hybridization. While this techniqueovercame some of the problems associated with the development of arapid, simple and versatile nucleic acid hybridization assay appropriatefor use in a clinical setting, the technique shared many of the samelimitations inherent in other hybridization procedures. For example,there remained an absolute limit on the extent of nucleic acid labelingby hybridization: at most, one complementary reporter probe can bind toeach accessible binding site on the target molecule. Furthermore, aswith other methods, hybridization rates and optimal hybridizationtemperatures depend on the target sequence's (G+C) content, a parameterwhich can vary considerably (discussed in detail below).

It is still another aspect of the present invention to provide reagentswhich do not depend upon the G+C content of target sequences as doconventional reagents.

When highly impure samples have been immobilized on filters, detectionhas been limited for the most part to radioactive methods (Bresser, J.,Doering, J. and Gillespie, D. (1983) DNA 2, 243-254; Fitts, R. (1985)Food Technology 39, 95-102). This presents a significant problem for aclinical laboratory, since many are not licensed to work withradioactive materials, some personnel have little or no experienceworking with radioactive materials, work with radioactive materialsinvolves some health risk, and radioactivity decays with time (in thecase of certain isotopes, quite rapidly) and can cause radiolysis ofprobes, thus limiting the usable lifetimes of these materials andreducing the feasibility of a clinical assay employing the materials.Non-radioactive detection methods have been developed (for example, seeLeary, J. J., Brigati, D. J. and Ward, D. C. (1983) Proc. Natl. Acad.Sci. U.S.A. 80, 4045-4049), but their sensitivity has not matched thatof the radioactive methods except where the target was highly purifiedprior to hybridization and detection.

`Sandwich hybridization` assays were developed (Dunn, A. R. and Hassell,J. A. (1977) Cell 12, 23-36; Ranki, M., Palva, A., Virtanen, M.,Laaksonen, M. and Soderlund, H. (1983) Gene 21, 77-85; Virtanen, M.,Palva, A., Laaksonen, M., Halonen, P., Soderlund, H. and Ranki, M.(1983) Lancet 1, 383-393) in an attempt to overcome some of thelimitations of conventional hybridization techniques. The target nucleicacid is `sandwiched` between a capture probe immobilized on a solidsupport and a labeled probe which is complexed with the target insolution. Though the `sandwich` technique represented an improvement insome aspects of nucleic acid hybridizations, the procedure stillretained certain weaknesses, notably its slowness, its clinicallyinsufficient sensitivity and its seemingly inherent background problemsthus lowering sensitivity and specificity.

Various techniques have been developed to accelerate the hybridizationprocess. For example the two-phase phenol/aqueous emulsion procedure(Kohne, D. E., Levinson, S. A., and Byers, M. J. (1977), Biochemistry16, 5329) has been reported to result in reaction rates over 100 timesfaster than reference rates. Deficiencies of this method, however, arethat it fails to boost reaction rates to the same extent when RNA isinvolved, that it requires that the reaction vessel be agitated duringthe hybridization process, and that it involves a chemical (phenol)associated with certain health risks.

It is still another aspect of the present invention to provide reagentswhich are effective in improving RNA hybridization rates.

It is still yet another aspect of the present invention to provideassays which overcome some of the deficiencies of conventional assays.

It is yet still another aspect of the present invention to providereagents which do not pose the health risks associated with phenol.

Another technique reported to accelerate hybridization reactions is toadd to a reaction mixture volume exclusion reagents, such aspolyethylene glycol (Renz and Kurz (1984), Nucl. Acids Res., 12,3435-3444), dextran or dextran sulfate (Wetmur, J. G. (1975),Biopolymers 14, 2517-2524; Wahl, G. M., Stern, M., and Stark, G. R.(1979), Proc. Natl. Acad. Sci. U.S.A. 76, 3683-3687; Wahl, G. M. andStark, G. R., U.S. Pat. No. 4,302,204, Nov. 24, 1981), polyacrylate orpolymethacrylate (Boguslawski, S. J. and Anderson, L. H. D., U.S. Pat.No. 4,689,294, Aug. 25, 1987). Though these methods acceleratehybridization when a target is immobilized the acceleration is lessdramatic in solution hybridizations. Furthermore, while these techniquesincrease hybridization rates, they do not raise final hybridizationlevels.

It is still further an aspect of the present invention to providereagents which increase hybridization rates in solution hybridization.

It is another still further aspect of the present invention to providereagents which raise final hybridization levels.

Kohne and Kacian (European Patent Application number 86304429.3) havealso reported a method of accelerating nucleic acid hybridization usingdifferent agents that also precipitate nucleic acids. In theirapplication, the claims are limited to increased rates of nucleic acidreassociation, with no mention whatsoever of increased levels (i.e.,final signals). There remains the labeling limit of, at most, onecomplementary reporter probe per binding site on the target molecule.Additionally, these investigators do not disclose any nucleic acidprecipitation agents which possess capabilities such as inactivatingnucleases, allowing proteases to function effectively, or strengtheningbinding, all which are further aspects to be provided by the reagents ofthe present invention.

Numerous modifications have been made to the original sandwichhybridization format, including replacing the filter with other solidsupports (Rashtchian, A., Eldredge, J., Ottaviani, M., Abbott, M., Mock,G., Lovern, D., Klinger, J. and Parsons, G. (1987) Clinical Chemistry33, 1526-1530); Langdale, J. A. and Malcolm, A. D. B. (1985) Gene 36,210-220) and the use of affinity methods to improve the speed of thecapture process (Rashtchian et al. (1987); Langdale and Malcolm (1985);Langer, P. R., Waldrop, A. A. and Ward, D. C. (1981) Proc. Natl. Acad.Sci. U.S.A. 78, 6633-6637; Manning, J., Pellegrini, M. and Davidson, N.(1977) Biochemistry 16, 1364-1370; Delius, H., van Heerikhuzen, H.,Clarke, J. and Koller, B. (1985) Nucl. Acids Res. 13, 5457-5469; Dale,R. M. K. and Ward, D. C. (1975) Biochemistry 14, 2458-2469; Banfalvi,G., Bhattacharya, S. and Sarkar, N. (1985) Anal. Biochem. 146, 64-70;Arsenyan, S. G., Avdonia, T. A., Laving, A. Saarma, M. and Kisselev, L.L. (1980) Gene 11, 97-108). These formats all employ a single capturestep. Insufficient specificity and sensitivity, however, remain problemsfor the clinical application of these techniques.

Another yet further aspect of the present invention is to provide assaysemploying new reagents which provide the necessary specificity andsensitivity for the clinical application of hybridization assays.

Another problem with the general application of conventionalhybridization techniques results from the fact that different targetnucleic acid sequences (and the corresponding complementary probes) varyconsiderably in (G+C) content. Consequently, since the hybrid meltingtemperature and the optimal hybridization temperature in commonly usedassay reagents vary as a function of the (G+C) content, they will bedifferent for target:probe combinations of different (G+C) composition(assuming the sequence lengths are the same). This means that theoptimal assay temperature in the standard assay reagents may bedifferent for each assay. This is highly undesirable since it makes itvery difficult to standardize the assay format to allow for the testingof many samples for different organisms in a common instrument at asingle temperature. Although probe length can be varied somewhat tocorrect for differences in melting temperatures (among probes withwidely differing (G+C) content), inclusivity and exclusivityrequirements set practical limits on this approach.

It is another aspect of the present invention to provide regents andmethods which employ a general assay protocol, e.g. temperature, for awide range of tests which is unavailable with hybridization media incommon use.

Melchior and von Hippel (Proc. Natl. Acad. Sci. U.S.A., 70 (1973),298-302) have observed that in solutions containing tetraalkylammoniumcations the A:T base-pair is strengthened relative to the G:C base-pair.Hamaguchi and Geiduschek (Jour. Amer. Chem. Soc. 84 (1962), 1329-1338)have reported that chaotropic anions denature nucleic acids and disruptthe A:T base pair more strongly than the G:C base-pair. Thesereferences, however, fail to disclose how the results may be utilized toprovide reagents meeting the various aspects of the present inventionand in particular fail to teach whether achieving GC/AT equivalence isdesirable and if so, how it is to be achieved.

It is yet another aspect to overcome the limit of one reporter probebinding to each target site inherent in all heretofore availablehybridization procedures.

It is still another aspect of the present invention to dramaticallyincrease the level of signals that one can obtain in traditionalhybridization assays.

All documents cited herein are fully incorporated herein by reference.

SUMMARY

In accordance with the various principles and aspects of the presentinvention there are provided herein novel organic salts andcombination-salts, having unique and useful properties related to thehybridization and detection of nucleic acids. Listed below are the mostuseful properties of the preferred hybridization reagents of the presentinvention. Preferred embodiments are those reagents which possess thefirst listed property (1), and additionally at least two (2) of theremaining eight (8) properties.

Each preferred reagent possesses the following property: 1) use of thereagents of the instant invention in hybridization assays results insignals of significantly greater magnitude than those obtained in commonreference reagents (SSC, SET, phosphate buffer or GuSCN). This observedphenomenon is referred to as hyperhybridization or superstoichiometriclabeling, reflecting the fact that more than one reporter molecule isbound per available binding site on the target molecule. We havesurprisingly discovered not only that the preferred reagents result inobservance of this phenomenon but also that the source of the elevatedsignals is something other than, or in addition to, simplehybridization. Accordingly, hyperhybridization should be understoodsimply as a reference to very high signals obtained in the hybridizationassay of the instant invention, and not as a statement that classicalhybridization is the source of the strong signals.

Additionally, each preferred reagent of the instant invention possessesat least two (2) of the following properties:

1) It minimizes the background noise which can result from eithernon-specific hybridization (of non-complementary or partiallycomplementary nucleic acids) or non-specific binding (for example, byelectrostatic forces) of labeled reporter probe to solid supports.

Alternatively stated, the Signal-to-Noise (S/N) ratio obtained in thereagent is at least 25% greater than that obtained in a common referencereagent, such as GuSCN.

2) It inactivates nucleases. Specifically, after a 15-minute incubationat 37° C. with up to 12.5 μg/ml RNase A in the reagent, at least 50% ofa population of target rRNA molecules remain intact, i.e., not cleavedby the RNase. This is especially useful in an assay involving nucleicacids, since nucleases could destroy targets and/or probes. Theintegrity of the nucleic acids is typically measured by theircapturability.

3) It allows proteases to function effectively. This may be convenientlymeasured by either of two assays. (1) Indirectly: in the assay describedin the immediately preceding paragraph, the addition of Proteinase K toa final concentration of 2.5 mg/ml results in there being at least 50%more intact rRNA than without the protease addition. (2) Directly: inthe reagent, after the addition of Proteinase K to a final concentrationof 2.5 mg/ml and a 10-minute incubation at 37° C., at least 50% of 5mg/ml BSA is solubilized. Protease activity is advantageous because itpotentially removes nuclease activity, helps disrupt target cells toexpose target molecules, and/or aids in the deproteinization of nucleicacids, which can in turn increase hybridization signals and lowerbackground signals.

4) It accelerates the speed of hybridization reactions by at least ≈50%relative to commonly used reference reagents (e.g. GuSCN).

5) It increases bond strengths of A:T base-pairs relative to G:Cbase-pairs such that the bond strengths of the two types of pairs areapproximately equivalent. This is ideally quantified by measuring thewidth of a nucleic acid melting curve, or by determining the meltingtemperatures of hybrids of different GC-content. Significant GC/ATequivalence is indicated, using probes approximately 35 nucleotides inlength, by a standard melting curve width of 5° C. or less, or by adifference of less than 10° C. between the dissociation temperatures ofprobes with 37%-GC and 66%-GC. If exact equivalence obtains, then itfollows that all probes of a given length will have approximately thesame melting temperature (T_(d)) in the reagent. A primary advantage ofthis is that hybridization conditions for a broad range of assays,employing probes of widely different GC contents, can be standardized(for example, in an automated format) because, for probes of a givenlength:

a) All hybridizations proceed at essentially the same rate at a giventemperature below T_(d) ;

b) All hybridizations proceed to essentially the same extent during afixed interval of time; and

c) All hybridizations are essentially equally stringent.

It will be understood that there may be modest differences in the rate,extent and stringency of hybridization as a function of the exactnucleotide sequence of each hybrid due to differences in more complexinteractions such as base stacking, interactions between chemicallymodified bases, etc.

6) It facilitates capture on solid supports, such as magnetic beads,advantageous for reversible target capture:

a) by promoting or accelerating the capture of poly-dA-tailedoligonucleotides on magnetic beads coated with oligo-dT. This isadvantageous in the most preferred hybridization assay in which thesolid support is oligo-dT-coated magnetic beads and in whichpoly-dA-tailed oligonucleotides, complementary to the desired target,serve as a bridge between the target and the solid support. This systemis described in greater detail in European Patent Application No.87309308.2; and

b) by preventing the aggregation of magnetic beads in various matrices.

7) It lyses cells to expose target nucleic acids.

8) It deproteinizes nucleic acids to make them accessible for efficienthybridization. This helps to control non-specific backgrounds, since wehave discovered protein-nucleic acid complexes are implicated innon-specific binding.

The novel organic salts of the present invention possess the first, andsome or all of the rest of the above properties and preferably compriseat least two ions: a tetraalkylammonium (TAA) cation and a chaotropicanion.

The anions employed in the preferred reagents of the first inventionideally are those which denature nucleic acids (i.e., which arechaotropic) and include for example: Trichloroacetate (TCA), Thiocyanate(SCN), Trifluoroacetate (TFA), Perchlorate (PCA), Iodide (I), andAcetate (CH₃ COO).

The cations employed in the preferred reagents of the present inventionideally are those which strengthen A:T base pairs, such as thetetramethylammonium (THA) or tetraethylammonium (TEA) ions.

Preferred embodiments of the present invention comprise TMA.TCA TMA.TFA,TMA.SCN:TMA.SCN (a 1:1 mixture, abbreviated "T³ ") and the mostpreferred embodiment, Guanidinium SCN (GuSCN):TMA.TFA (a `family` ofmixtures comprising these two salts in various relative concentrationratios, abbreviated "GT³ ").

GT³ is most preferred because it advantageously comprises the followingcharacteristics:

1) it promotes superstoichiometric labeling;

2) if effects approximate GC/AT equivalence (Evaluations of GC/ATequivalence are based on two different types of information: (a) thesharpness of a melting curve(s) (the sharper the curve, the greater thedegree of equivalence) and (b) the closeness of melting temperature(Td's) of hybrids of different GC content (the closer the Td's, thegreater the degree of equivalence));

3) it inactivates nuclease (RNase A);

4) it minimizes background/specificity problems;

5) it fosters efficient target capture on magnetic beads;

6) it accelerates hybridization relative to conventional reagents;

7) it is stable at room temperature and 37° C. for at least six months;and

8) with respect to its ability to lyse cells, the most preferred assaymethod comprises first lysing the target cells in 5M GuSCN, part of theGT³ composition which is then advantageously completed by adding anequal volume of 4M TMA.TFA in which hybridization ideally takes place.Since GuSCN is an efficient lysis agent and GT³ is a superiorhybridization agent, such a protocol results in efficient lysis followedby superior hybridization.

Other preferred embodiments possess certain of the aforementioneddesirable capabilities and accordingly each has clear utility in and ofitself. (For example, a reagent that denatures nucleic acids but notproteins is ideally used in enzymatic amplification reactions.) Inapproximate order of decreasing preference they are: T³, TMA.SCN,TMA.TFA, and TMA.TCA, all of which are still superior to standardhybridization solutions (SET, SCC, Phosphate or GuSCN) which have beenused as reference. For example, GuSCN does not effectsuperstoichiometric labeling, does not accelerate hybridization tonearly the same extent, does not bring about GC/AT equivalence, and isnot as suitable as a medium for bead capture since it requires greaterdilution and causes some aggregation; SSC, SET, and Phosphate do notbring about superstoichiometric labeling, do not inactivate nuclease, dolittle to accelerate hybridization, do not equalize GC/AT base pairs,cause bead clumping, do not lyse cells and only moderately deproteinizenucleic acids.

A most preferred detection method suitable for use on an automatedsystem comprises:

a) combining the sample to be analyzed with an entity which disruptsmolecular structure, mixing thoroughly, and incubating at an appropriatetemperature for an appropriate length of time, thereby exposing a targetnucleic acid;

b) adding a specific capture probe, mixing thoroughly, and incubating ata temperature and length of time to permit hybridization of said probeto the target nucleic acid;

c) adding a solid support, mixing thoroughly, and incubating at anappropriate temperature for an appropriate length of time, so as tocapture the hybrids on said solid support;

d) physically separating any uncaptured materials from the solidsupport;

e) adding a wash buffer, mixing thoroughly, and physically separatingany unbound materials from the solid support; and repeating this step asoften as desired;

f) adding to the solid support having attached targets a release buffer,mixing thoroughly, and incubating at an appropriate temperature for anappropriate length of time so as to cause said targets to be releasedfrom said solid support;

g) removing from the solid support the solution which contains thereleased targets and transferring said solution to a separate vessel;

h) adding a labeled (reporter) probe, mixing thoroughly, and incubatingat an appropriate temperature for an appropriate length of time wherebyhybridization of the reporter probe to the target can occur;

i) adding an entity which will effect superstoichiometric labeling,mixing thoroughly, and incubating at an appropriate temperature for anappropriate length of time;

j) adding a solid support, mixing thoroughly, and incubating at anappropriate temperature for an appropriate length of time, so as tocapture the hybrids on said solid support;

k) physically separating any uncaptured materials from the solidsupport;

l) adding a wash buffer, mixing thoroughly, and physically separatingany unbound materials from the solid support; and repeating this step asoften as desired;

m) adding to the solid support with attached targets a release buffer,mixing thoroughly, and incubating at an appropriate temperature for anappropriate length of time, so as to cause said targets to be releasedfrom said solid support;

n) removing from the solid support the solution which contains thereleased targets, and transferring said solution to a separate vessel;

o) carrying out one or more additional capture/release cycle(s) (steps hthrough 1) to reduce background (noise) levels;

p) optionally, dissolving the precipitates (which contain the targetsand probes) and rendering the labeled probes monodisperse so that theyare all accessible for detection;

q) adding appropriate chemical and/or biological reagents,simultaneously or sequentially, mixing thoroughly, and incubating at anappropriate temperature(s) for an appropriate length(s) of time so as todetect the superstoichiometrically labeled targets.

It will be recognized by those skilled in the art that various steps ofthe preceding procedure may be conveniently combined.

Further understanding may be had concerning the various properties ofthe preferred embodiments of the present invention by reference to thedetailed Description.

GLOSSARY Glossary of Abbreviations, Terms, and Probe Sequences Used:

EDTA--Ethylenediaminetetraacetic acid

Gu--Guanidinium

GuSCN or GT--Guanidinium thiocyanate

TCA--Trichloroacetate

TEA--Tetraethylammonium

TFA--Trifluoroacetate

TMA--Tetramethyammonium

TMA TCA--Tetramethyammonium:Trichloroacetate (this reagent can compriseany of a variety of concentrations of these ions; when not otherwisenoted, the two ions are present in equimolar amounts at approximately3M; ordinarily buffered with phosphate and borate, and/or EDTA)

TMA.TFA--Tetramethyammonium:Trifluoroacetate (this reagent can compriseany of a variety of concentrations of these ions; when not otherwisenoted, the two ions are present in equimolar amounts at approximately4M; ordinarily buffered with phosphate, borate and EDTA)

TMA.SCN--Tetramethyammonium:Thiocyanate (this reagent can comprise anyof a variety of concentrations of these ions; when not otherwise noted,the two ions are present in equimolar amounts at approximately 5-6M;ordinarily buffered with phosphate, borate and EDTA)

T³ --TMA.TFA:TMA.SCN (a family of reagents comprising solutions ofvarious concentrations of these two compounds; when not otherwise noted,a 1:1 mixture of TMA.TFA and TMA.SCN resulting in final concentrationsof approximately 2M TMA.TFA and 3M TMA.SCN)

GT³ --GT:TMA.TFA (a family of reagents comprising solutions of variousconcentrations of these two compounds; when not otherwise noted, a 1:1mixture of GT and TMA.TFA resulting in final concentrations ofapproximately 2.5M GT and 2M TMA.TFA)

GT³ *--GT:TMA.TFA (a member of the GT³ family of reagents; unlessotherwise noted, a 2:3 mixture of GT and TMA.TFA resulting in finalconcentrations of approximately 2M GT and 2.4M TMA.TFA)

GT⁵ --GT:TMA.TFA:TMA.SCN (a member of the GT³ family of reagents; unlessotherwise noted, a 1:1:1 mixture of the three substituent reagentsresulting in final concentrations of approximately 1.7M Gu, 3.7M SCN,3.3M TMA and 1.3M TFA)

T_(d) --Dissociation (melting) temperature.

.sup.Δ T_(3/4-1/4) --Melting curve width

RTC--Reversible Target Capture

Hybridization--The highly specific association of two mutuallycomplementary single-stranded nucleic acid molecules.

Dissociation (used interchangeably with melting temperature)

temperature--The temperature at which 50% of the nucleic acid hybridsinitially present in a population are dissociated.

Melting curve width--By convention, the difference between thetemperature at which 75% of a population of pre-formed, meltable hybridsremain associated and that at which 25% are still associated.

Chaotrope--Large anions which, at high concentrations, or in some casesat low pH, disrupt the water lattice and thereby the structure ofnucleic acids (lowering their dissociation temperature) and/or proteins(lowering their transition temperature).

Capture probe--A nucleic acid molecule comprising two moieties: one partwhich can hybridize to a target molecule, and an attached part which canbind to a solid support. (In the preferred assay of the presentinvention the latter part is a poly-dA `tail` which can bind reversiblyto oligo-dT-coated magnetic particles.)

Reporter probe--A nucleic acid molecule `labeled` with a detectableentity (e.g. fluorophere, radioactive isotope or an antigen to which anenzyme-antibody conjugate can bind) which can hybridize to a targetmolecule.

Riboprobe--A single-stranded RNA molecule typically made by in vitrotranscription (such as by using SP6 or T7 RNA Polymerase) of a portionof a vector containing the appropriate DNA segment. The preferredriboprobes used herein are exactly complementary to either the 3'- or5'-half of E. coli 16S ribosomal RNA and are able to hybridize with alleubacterial rRNAs tested by virtue of several universally conservedsequences in the 16s rRNAs of eubacteria, (the prefix in the designationof the Riboprobe indicates the portion of the rRNA to which it iscomplementary), and are labeled with 32P, biotin or fluorescein.

BioRiboprobe--A Riboprobe containing biotinylated nucleotides.

FluoroRiboprobe--A Riboprobe containing fluorosceinated nucleotides.

Reversible Target Capture--A process in which target nucleic acids arepurified by sequential cycles of capture on and release from varioussolid supports as disclosed in detail in European Patent Application No.87309308.2.

Hyperhybridization--The phenomenon of a hybridization assay giving riseto signals of greater magnitude than usually observed in a referencereagent such as GuSCN or 6x SET

Superstoichiometric labeling--A more precise and accurate term todescribe "hyperhybridization," reflecting the fact that the`hyper-signals` are due to a type of binding other than truehybridization (probably co-precipitation) and that more than one labeledprobe is bound per accessible target site.

    __________________________________________________________________________    Probe                                                                             Target                                                                             Sequence (5'→3')                                              __________________________________________________________________________    R444                                                                              E. coli                                                                            AAT GAG CAA AGG TAT TAA CTT TAC TCC CTT CCT CCC C                     666                                                                              List.                                                                              TGT CCC CGA AGG GAA AGC TCT GTC TCC AGA GTG GTC AAA GAT AT            676                                                                              Salmon.                                                                            TCA ATT GCT GCG GTT ATT AAC CAC AAC ACC TTC CT                        730                                                                              E. coli                                                                            TAA CGT CAA TGA GCA AAG GTA TTA ACT TTA CTC CC                        732                                                                              Campy.                                                                             TCC AAC TGT TGT CCT CTT GTG TAG GGC AGA TTA AC                        733                                                                              Campy.                                                                             TGT GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC GT                        787                                                                              E. coli                                                                            TCA ATG AGC AAA GGT ATT AAC TTT ACT CCC TTC CT                        853                                                                              E. coli                                                                            ACG GTC CAG ACT CCT ACG GGA GGC AGC AGT GGG GA                        854                                                                              E. coli                                                                            TCC CCA CTG CTG CCT CCC GTA GGA GTC TGG ACC GTA TAT                   855                                                                              E. coli                                                                            GGG AGT AAA GTT AAT ACC TTT GCT CAT TGA CGT TA                       1009                                                                              Campy.                                                                             C*AT TCA CCG TAG CAT GGC TGA TCT ACG ATT ACT C*T                     1010                                                                              Campy.                                                                             C*CC GAA CTG GGA CAT ATT TTA TAG ATT TGC TCC C*T                     __________________________________________________________________________     C* indicates a modified Cytidine residue                                 

BRIEF DESCRIPTION OF THE DRAWINGS

Further understanding of the various aspects and principles of thepresent invention may be had by reference to the drawings wherein:

FIG. 1 shows hybridization in GT³ as a function of temperature;

FIG. 2 graphically compares hybridization in GT³ with a variety ofcontrols;

FIG. 3 graphically depicts hybridization in GT³ as a function of captureprobe concentration;

FIG. 4 graphically depicts the interplay of various probes in apreferred assay;

FIG. 5 graphically depicts RNase A activity of various reagentembodiments of the present invention.

I. GUSCN:TMA.TFA("GT³ ")

A reagent containing equal parts of 5M GuSCN and 4M TMA.TFA (hereinafterabbreviated as "GT³ ") is the most preferred reagent embodiment of thepresent invention. It provides excellent RNase-control characteristicsand support of efficient and specific hybridization including"superstoichiometric labeling": the binding of more than one detectablemolecule of probe to each target site on each target molecule. Indeed,the experiments described below demonstrate that GT³ meets all of theobjectives in terms of desired properties previously listed.

This reagent advantageously utilizes in synergy the properties of itsindividual components: GuSCN, known to have desirable RNase-inhibitingproperties; and TMA.TFA, which was found to possess the ability toaccelerate and enhance hybridization (section 5). It was surprisinglyfound that GT³ possesses all the properties of its individualcomponents, contrary for example to the less preferred TMA.TFA.

A. General Methods

In the various experiments described herein, the following generalprotocols were followed. Variations from these procedures are describedin those experiments where they occur.

DNA:RNA Hybridization

1) The following were combined: reagent, Dextran Sulfate (to a finalconcentration of 10%), rRNA target (cell extracts at a definedconcentration) and reporter probe (³² P-labeled 3'-Riboprobe). Themixture was incubated to bind the reporter probes to the target rRNA.

2) The capture probe (dA-tailed oligomer) (see European Appliction No.87309308.2) was added and the mixture was incubated at the desiredtemperature.

3) Aliquots were removed at defined times and added to oligo-dT-coated1μ magnetic beads obtained from Advanced Magnetics pre-warmed to 37° C.if their volume was equal to or greater than that of the hybridizationmixture. A small volume (e.g. equal to or slightly less than the volumeof the sample aliquot) of concentrated beads was used for capture in TMAsalts, while a large volume (e.g. twice that of the aliquot) of moredilute beads was used for capture in GuSCN. The resulting mixture wasincubated for three minutes to capture the hybrids on the beads.

4) The beads were separated magnetically using a magnetic separator suchas is commercially available from Corning or that described in copendingU.S. Ser. No. 121,191 and the supernatant removed. The beads were washedtwice with wash buffer between 0.5M or 2M GuSCN. The beads wereresuspended in wash buffer and the amount of radioactivity bound to thebeads was determined by a scintillation counter.

5) Using the specific activity cpm/fmole! of the Riboprobe, the numberof Riboprobes captured was calculated. Then, knowing the number of cellsin the sample, the number of riboprobes captured per cell wascalculated. (Prior to use in these experiments, the concentration(cells/ml) of each freshly grown cell preparation was determined byserial dilution, growth on solid media and colony counting. Solutions ofcells were then combined with GuSCN (to a final 2.5M) and stored at -20°C. until needed for experiments.)

At the outset of this work, it was assumed that one Riboprobe was boundto each molecule of 16S rRNA, implying a 1:1 Riboprobe:ribosome ratio.However, in a variety of experiments abnormally high numbers ofriboprobes per cell were seen (e.g., significantly greater than theaccepted norm of 10,000-20,000 ribosomes/cell in Salmonella). As will bediscussed below, this was discovered to be due to the fact more than oneRiboprobe was bound to each molecule of 16S rRNA. To reflect thisdiscovery, "Riboprobes captured/bound per cell" are reported hereinrather than "Ribosomes bound per cell."

Melting and elution studies

1) Reagent for the present invention (herefter "reagent") DextranSulfate (to a final concentration of 10% by weight), nucleic acid target(rRNA from cell extracts at a defined concentration, or an artificialtarget comprising a ³² P-labeled oligomer), and if the target was rRNA,a ³² P-labeled 3'-Riboprobe, and a capture probe (dA-tailed oligomer)were combined, and the mixture incubated for at least an hour at 37° C.to allow hybridization to occur.

2) The mixture was diluted 1:10 with the reagent (without DextranSulfate), and held at the hybridization temperature for the remainder ofthe experiment.

3) Approximately 0.20 ml Aliquots were removed from the mixture andincubated at various elevated temperatures, e.g. 40° C. to 95° C., forsix (6) minutes.

4) To each heated aliquot, immediately after the six-minute incubation,and periodically to aliquots taken from the constant-temperature mixture(No. 2 above) 10 μl magnetic beads were added at a concentration ofabout 100 O.D. 550/ml. The mixtures were incubated at the designatedtemperatures, and the beads were separated, washed and counted as above(#3 & #4). The constant-temperature-mixture measurements served as abaseline to make sure that the level of hybridization was not changingwith time. At each elevated temperature, the amount of hybridized targetnucleic acid, relative to the constant-temperature reference, wasdetermined.

5) For elution experiments, a 200 μl aliquot was taken from theconstant-temperature mixture, 10 μl of heads were added and incubated,the beads were separated magnetically, and the supernatant was removed.An equal volume of 2.5M GuSCN was added to the beads and the mixture wasincubated at 37° C. for 5 min. The beads were separated magnetically andthe supernatant was removed. The beads were resuspended in wash buffer,the beads and the supernatant were counted in a scintillation counter,and the percent elution was calculated.

B. Bead capture

Experiments to be described below demonstrated that when hybridizationwas carried out in TMA.TFA, capture by oligo-dT-coated magnetic beadswas possible without any dilution of the reagent: i.e., simply by addinga small volume of concentrated beads (much smaller than the volume ofthe hybridization mixture) and incubating. This was a very advantageousproperty of the reagent, and an unexpected improvement over GuSCN whichmust be diluted three-fold for capture. To determine whether capturewould take place in GT³ without dilution, or how much dilution would benecessary, target and probes were hybridized to completion in GT³, afterwhich aliquots of the hybridization mixture were taken and combined withvarious volumes of oligo-dT-coated magnetic beads of variousconcentrations (such that the total amount of beads was the same in eachcase). After three-minute incubations at 37° C., the beads were washedtwice and counted.

Table 1 presents the averaged results from three separate experiments,listing the amount of hybrids captured with various volumes of beads,relative to the amount captured with two volumes of beads ("one volume"means a volume equal to the volume of the hybridization mixturealiquot); numbers in parentheses are standard errors of the data.

                  TABLE 1                                                         ______________________________________                                        Bead capture in GT.sup.3                                                      Bead volume                                                                              Relative amount of complex captured                                ______________________________________                                          2×  100%!                                                               1× 93% (+/-8%)                                                        0.5× 55% (+/-15%)                                                       0.25×                                                                              39% (+/-16%)                                                       ______________________________________                                    

These results show that with this reagent one need add only one volumeof beads to one volume of hybridization mixture (i.e., dilute thereagent by 1/2) in order to capture complexes efficiently.

C. Proteinase K activity

Proteinase K (PK) is moderately active in, while ribonuclease issignificantly inhibited by, GuSCN. Prior to empirical determination, itcould not be predicted whether addition of the GuSCN to TMA.TFA wouldproduce a reagent with the capability of controlling nucleases. Theextent of Proteinase K activity in GT³ was assayed by determining itsability to digest (solubilize) ¹⁴ C-labeled bovine serum albumin (BSA).

The results of our experiments are presented in Table 2 below. Theseexperiments were done twice, and the percentages below are the averagesof the two experiments; the numbers in parentheses are the experimentalvariations.

                  TABLE 2                                                         ______________________________________                                        Proteinase K activity in various reagents                                     Reagent     % BSA solubilized                                                 ______________________________________                                        GuSCN       41% (+/-10%)                                                      GT.sup.3    92% (+/-1%)                                                       ______________________________________                                    

The results indicate a high level of PK activity observed with GT³. Inanother experiment it was observed that approximately 40% of the initialProteinase K activity remained after incubation of the enzyme in GT³ at37° C. for two hours. This contrasts favorably with a similar incubationin GuSCN, after which only 10% of the initial activity remained. Thisdemonstrates a clear and unpredictable advantage of GT³ over GuSCN,since whenever lengthy digestions are required for efficient cell lysisand deproteinization, the reagent in which Proteinase K is more stablewill be more advantageous.

Subsequent experiments showed that Proteinase K is similarly active in arelated reagent, GT⁵, a 1:1:1 mixture of 5M GuSCN, 4M TMA.TFA and 6MTMA.SCN. It has also been observed that the level of Proteinase Kactivity gradually decays upon incubation at 37° C. in this reagent,with a half-life of approximately 2 hours. Again, this is a significantadvantage over GuSCN, in which the half-life is less than an hour.

D. Ribonuclease activity

Though the ability of a hybridization medium to support proteaseactivity is desirable, an equally (or more) important property is thecapability of inhibiting nucleases, especially ribonucleases. In thissection experimental evidence is presented indicating that GT³ and GT⁵both inhibit the activity of a typical ribonuclease, RNase A. In thefirst experiment reported in this section, in addition to assayingnuclease activity in GT³ and in GT⁵, a full set of control reactions wasperformed in GuSCN (an established inhibitor) and phosphate buffer (inwhich the enzyme is known to be active). The experimental protocol was afollows:

1) A target complex was prepared by hybridizing target rRNA (fromSalmonella extracts), capture probe (dA-676), and ³² P-labeled3'-Riboprobe (as earlier described).

2) The target complex was diluted into each of the above four testreagents.

3) RNase A solutions of various concentrations (0-250 μg/ml in 0.15Mphosphate buffer pH7) were prepared.

Into tubes of the diluted target complex was added RNase A, to one ofthe final concentrations noted in Table 3 below. The control comprisedonly phosphate buffer diluent. The mixtures were incubated at 37° C. for15 minutes. Proteinase K was added to a final concentration of 1.25mg/ml to stop the nuclease activity.

4) Tailed probes and hybridized complexes were captured on magneticbeads (as previously described), which were washed and counted. Thelevel of RNase inhibition was calculated as follows. ##EQU1##

                  TABLE 3                                                         ______________________________________                                                  RNase A inhibition in various reagents                               RNase A! % RNase inhibition                                                  ug/ml     Phosphate                                                                              GuSCN       GT.sup.3                                                                           GT.sup.5                                  ______________________________________                                        0.00       100%    100%        100% 100%                                      0.13      0.84%    100%        100% 100%                                      1.25      0.05%    100%        100% 100%                                      4.17      0.52%    100%        100% 100%                                      12.50     0.06%    100%        100%  93%                                      ______________________________________                                    

These data demonstrate that GT³ exhibits potent inhibition of RNase A.Additionally, this experiment demonstrates that GT⁵ also possessesnearly the same RNase inhibitory capabilities.

The data also show that there is considerable flexibility in adjustingthe relative concentrations of various components of the reagent(s) inorder to retain the desired nuclease control.

E. Hybridization in GT³

Since there was no certainty that hybridization would take placeefficiently in GT³, experiments were undertaken to so determine.

1) Hybridization temperature optimization

In this section, data are reported from experiments to determine whichof three potential reaction temperatures (room (22°-24° C.), 37° C. and45° C.) best promoted hybridization. In these experiments, the targetwas rRNA from Salmonella extracts at 9×10⁶ cells/ml, the reporter probewas a 32P-labeled 3'-Riboprobe at 1 μg/ml, and the capture probe wasdA-tailed oligo 676 (see U.S. Ser. No. 127,484 for further details) at0.5 μg/ml. Target and riboprobe were incubated at 37° C. for 4 hours toprehybridize them, after which various aliquots of the mixture wereeither kept at the same temperature or re-equilibrated at one of theother temperatures. Capture probe was then added to each, timedincubation was begun and aliquots were removed after 5, 15, 30 and 60minutes. The removed aliquots were added to an equal volume of magneticbeads (pre-warmed to 37° C.), mixed and incubated at 37° C. for threeminutes to capture the hybridized complexes. The beads were then washedtwice and counted. From the number of cpm captured, the specificactivity of the riboprobe and the cell concentration, the number ofriboprobes captured per cell was calculated for each time/temperaturepoint. Three controls were done at each temperature: omitting thecapture probe, omitting target cells/rRNA and substituting heterologouscells (E. coli) for the true target. The results of these experimentsare presented in FIG. 1.

From these plots, 37° C. appears to be the optimized hybridizationtemperature followed closely by room temperature. This is especiallyadvantageous since 37° C. is optimal for an instrument-based assay. Thedata also show that temperature between room temperature and 37° C. maybe alternately preferred. This embodiment therefore providesconsiderable flexible in designing and performing a hybridization assay.

Further, the numbers of labeled probes captured per cell (>100,000) inthis experiment are exceptionally high. Apparently the reagent assiststhe capture of approximately 10 riboprobes per 16S rRNA molecule targetin these cells.

2) Hybridization in GT³ and other reagents

For these experiments, target rRNA (from various cell extracts) wasincubated for three hours with 1 μg/ml ³² P-labeled 3'-Riboprobe(selected in accordance with the target rRNA as set forth in theGlossary) in the reagent of interest with 10% (mass/volume) DextranSulfate at 37° C. The dA-tailed capture probe was then added to a finalconcentration of 0.25-0.50 μg/ml, and the mixture was incubated at 37°C. Finally, the hybridization complex was captured on magnetic beads(two volumes of beads per volume of hybridization mixture), washed, andthe extent of hybridization determined by scintillation counting. Forall experiments, three controls were done: (1) a reaction in which thetarget rRNA was omitted, (2) a reaction in which the capture probe wasomitted, and (3) a reaction in which heterologous cells (cells otherthan those for which the capture probe was specific) were substitutedfor the experimental target cells. Various reagents were tested, aslisted below.

The results from these experiments are presented in Table 4. The numberof riboprobes bound per cell was calculated from the following known ordetermined quantities: cpm/fmole of riboprobe, cells per reaction, andcpm captured per reaction. For each control, the captured "noise" as afraction of "signal" was calculated.

                  TABLE 4                                                         ______________________________________                                        Hybridization in various reagents                                             ______________________________________                                        Target cells: Salmonella                                                                              Campylobacter                                                                            E. coli                                                  8.5 × 10.sup.6 /ml                                                                1 × 10.sup.7 /ml                                                                   8 × 10.sup.6 /ml                     Capture Probe:                                                                              dA-676    dA-732     R444                                                     0.5 ug/ml 0.5 ug/ml  0.25 ug/ml                                 Control cells:                                                                              E. coli   E. coli    Salmonella                                               8 × 10.sup.7 /ml                                                                  8 × 10.sup.7 /ml                                                                   8.5 × 10.sup.6 /ml                   Reagent                                                                       GuSCN   Ribos./cell:                                                                            1,000     500      2,100                                            N/S,no trg.:                                                                            56%       54%       6%                                              N/S,no prb.:                                                                            78%       63%      12%                                              N/S,het.  83%       62%       8%                                              cells:                                                                75% GuSCN                                                                             Ribos./cell:                                                                            900                                                         25%     N/S,no trg.:                                                                            33%                                                         TMA.TFA N/S,no prb.:                                                                            33%                                                                 N/S,het.  56%                                                                 cells:                                                                50% GuSCN                                                                             Ribos./cell:                                                                            220,000   90,000   130,000                                  50%     N/S,no trg.:                                                                             5%        4%      10%                                      TMA.TFA N/S,no prb.:                                                                            0.2%      0.1%      .5%                                     "GT.sup.3 "                                                                           N/S,het.   2%        4%      23%                                              cells:                                                                25% GuSCN                                                                             Ribos./cell:                                                                            130,000                                                     75%     N/S,no trg.:                                                                            79%                                                         TMA.TFA N/S,no prb.:                                                                            0.4%                                                                N/S,het.   6%                                                                 cells:                                                                TMA.TFA Ribos./cell:                                                                            170,000                                                             N/S,no trg.:                                                                            79%                                                                 N/S,no prb.:                                                                            0.3%                                                                N/S,het.   4%                                                                 cells:                                                                ______________________________________                                    

These results show that GT³ is the most preferred hybridization reagent.Additional observations are:

a)Hybridization in GT³ is significantly superior to that in GuSCN. Thenumber of riboprobes captured per cell is exceptionally high.

b)The backgrounds are almost all very low in GT³, demonstrating thespecificity of the hybridization. The favorable background control is afeature that distinguishes the GT³ (50:50 mixture) from reagent mixtureswith a higher concentration of TMA.TFA.

The experiments just discussed indicated that as a hybridization reagentGT³ was clearly superior to GuSCN. Those experiments were repeated forT³ (GT³ without GUSCN), GT⁵ (a 1:1:1 mixture of 5M GuSCN, 4M TMA.TFA and6M TMA.SCN). The experimental protocol was exactly as above except: theincubation with target and riboprobe only was done overnight rather thanfor four hours, and the incubations were at 37° C. in GT³ and GT⁵ and at45° C. in T³. The results of these experiments showed that the rate andthe extent of hybridization were greater in GT³ than in T³ or GT⁵.

3) Signal-to-noise (S/N) ratios

The following experiment demonstrates that not only does one obtainstrong signals in GT³, but also at an advantageous S/N ratio. Theprotocol was exactly as above (37° C. incubations, with a 3 1/2-hourtarget+riboprobe pre-incubation). The results obtained are presented inFIG. 2. The controls were: capture probe omitted (+), target cellsomitted (diamond), and heterologous (E. coli) rRNA substituted for thetrue target (Salmonella) rRNA (triangle). For reference, 300,000 cpmcorresponds to approximately 172,000 riboprobes bound per cell.

As can be seen in FIG. 2, the controls were, as hoped for, quite lowrelative to the signal. In this experiment, the "no cell" and"heterologous cell" controls gave noise at 0.7-1.3% of the signal andthe "no probe" control gave noise at 0.07-0.5% of the signal.

4) Reversible Target Capture and Signal-to-Noise Enhancement

The reduction of background levels is essential if one wishes to fullyexploit the hyperhybridization potential of the new reagent(s). One wayto reduce backgrounds is to take advantage of Reversible Target Capture(RTC): after the first capture, to elute (with 2.5M GuSCN, at 37° C.)and recapture (with two volumes of beads at 37° C.) the probe-targetcomplexes, repeating this cycle as often as necessary to achieve thedesired S/N ratio. The utility of this assay procedure was evaluated inthe following experiment.

Protocol for Hybridization and Elution/Recapture

Hybridizations were performed using the generic eubacterial5'-riboprobe, dA-tailed oligonucleotide #666 and either E. coli alone(10⁸ cells/ml; control) or E. coli (E.C.) (10⁸ /ml) and Listeriamonocytogenes (L.M.) (3×10⁶ cells/ml). Cell extracts had been preparedby lysis with lysozyme, mutanolysin and Proteinase K. Hybridization wasfor 15 minutes at 37° C. Prewarmed magnetic beads (binding capacity: 1μg/ml dA₁₂) were added (one volume for GT³ captures and two volumes forGT captures) and capture was effected by incubation at 37° C. for 3minutes. After magnetic separation, the beads were washed three times,and then the hybrids were eluted by adding one volume of 2.5M GuSCN andincubating for 1 minute at 37° C. For recapture, two volumes of beadswere added and the mixture was incubated for 3 minutes at 37° C. Thefirst and second sets of beads were scintillation-counted, as was thematerial which was eluted from the first set of beads but did not rebindto the second set. For each sample, the number of counts initially boundto the first set of beads was calculated by adding the final countsstill bound to both sets of beads, and the counts eluted from the firstset but not bound to the second. Similarly, the total counts eluted fromthe first set of beads were calculated by adding the un-re-bound elutecounts and the counts on the second set of beads. The data from thisexperiment are presented in Table 5

                  TABLE 5                                                         ______________________________________                                        Hybridization and Elution/Recapture in GT and GT.sup.3                                 GuSCN   GuSCN    GT.sup.3  GT.sup.3                                           Signal  Noise    Signal    Noise                                              (L.m. + E.c.)                                                                         (E.c.)   (L.m. + E.c.)                                                                           (E.c.)                                    ______________________________________                                        Counts initially                                                                         23,000    11,100   168,900 10,900                                  bound to first set                                                            of beads                                                                      Counts eluted from                                                                       15,600    510      105,800 700                                     first set of beads                                                            Counts bound to                                                                          4,500     30       87,200  45                                      second set of beads                                                           ______________________________________                                    

These results clearly demonstrate the advantage gained withelution/recapture. In GT³, for example, the Signal-to-Noise ratioincreased from 15:1 after the first capture to almost 2000:1 after thesecond capture. This illustrates how assays in GT³ can be modified tomost fully capitalize on the hyperhybridization phenomenon. It is worthnoting that the S/N ratios in GT³ after one and after two rounds ofcapture are more than ten times better than with conventionhybridization in GuSCN.

Without wishing to be bound thereby it is our hypothesis that the basisof GT³ hyperhybridization is RNA precipitation. Assuming that this wasthe underlying mechanism, a further modification was made to the assayto circumvent non-specific precipitation of competitor molecules inbiological samples. The following protocol reflects this modification.

Since large quantities of RNA may be present in biological specimens andsince GT³ can precipitate RNA, the targets and capture probes were firsthybridized in GuSCN, and captured on the solid support. Then, thetarget-probe complex was washed and eluted (in 2.5M GuSCN) from thesolid support, 4M TMA-TFA and riboprobe were added (the solution thenapproximated GT³), and the mixture was incubated. The intent was toproduce strong signals (due to hyperhybridization) and minimalbackgrounds. Specific experimental details were as follows:

Campylobacter jejuni (final concentration: 10⁷ cells/ml) and E. coli(final concentration: 10⁸ /ml) extracts were combined in either 2.5MGuSCN (GT) or GT³. Control experiments contained no Campylobacterextracts. In the first round of hybridization and capture, twoCampylobacter-specific oligonucleotide probes were used (see U.S. Ser.No. 216,679): ³² -labeled oligonucleotide #732 and dA-tailed #733. (Foran illustration of the probe reactivity scheme, see FIG. 5 anddiscussion pertaining thereto.) The probe-target complexes were elutedfrom the beads with 2.5M GuSCN. An aliquot of the eluate wasscintillation-counted. Next, the eluted complexes were hybridized with³² P-labeled 3'-Riboprobe in either GT or GT³. Finally, the hybrids werecaptured on a second set of beads, and the beads washed and counted. Thenumber of probes captured per cell can be calculated from the cellconcentration and the specific activity of the labeled probe. Theresults are presented in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    Preliminary target:DNA-probe hybridization in GT or GT.sup.3                  followed by target:RNA-probe hybridization in GT or GT.sup.3                  First round:          Second Round:                                           Reporter probe = oligonucleotide                                                                    Reporter probe = riboprobe                                  Sig. Noise                                                                             Probes       Sig.  Noise                                                                              Probes                                   Reag.                                                                             (cpm)                                                                              (cpm)                                                                             per cell                                                                           S/N Reag.                                                                             (cpm) (cpm)                                                                              per cell                                                                           S/N                                 __________________________________________________________________________    GT  11,600                                                                             5,600                                                                             4,600                                                                              2:1 GT  32,600                                                                              2,100                                                                              530  16:1                                                      GT.sup.3                                                                          1,200,000                                                                           10,300                                                                             19,500                                                                             120:1                               GT.sup.3                                                                          16,400                                                                             2,200                                                                             6,500                                                                              7:1 GT  92,000                                                                              2,300                                                                              1,500                                                                              40:1                                                      GT.sup.3                                                                          2,130,000                                                                           37,900                                                                             34,700                                                                             56:1                                __________________________________________________________________________

These data support the following:

a) The first hybridization of an assay may be advantageously performedin GuSCN provided it is followed by a second in GT³, withhyperhybridization taking place in the second step.

b) Hyperhybridization did indeed take place in a reagent formed by theaddition of TM A.TFA to GuSCN after chemical elution in the latter.(This medium is similar in composition to 1:1 GT³ and belongs to thefamily of GT³ reagents.)

c) The fact that hyperhybridization occurred after the probe-targetcomplex was purified from cellular debris by a round of target captureindicates that it does not require high concentrations of cellularcomponents such as proteins, etc.

d) Hyperhybridization took place in GT³ when the labeled probe was anRNA molecule (riboprobe), but not when it was a short (DNA)oligonucleotide. (Compare Round 1 and Round 2 numbers.) Campylobactercells were estimated to contain approximately 5,000 ribosomes each. Thenumber of probes captured per cell in GT³ in the first round (with a DNAlabeled probe) was not very much greater than this or the same number inGT. The number of probes per cell in GT³ in the second round (with anRNA labeled probe) was considerably greater than this, indicatinghyperhybridization).

e) Not only were the signals stronger when hybridization took place inGT³ with a labeled RNA probe (i.e. when there was hyperhybridization),but the S/N ratios were also greater. (Compare to the results presentedin Table 5).

5) Capture probe concentration

It is generally desirable to know how much capture probe is preferablyused in order to capture the majority of available targets in a shortperiod of time. If, as was found to be the case in other TMA reagents,acceptably efficient hybridization takes place at much lower probeconcentrations than in GuSCN, there is the potential of significantlyreducing the cost of the assay. To determine whether this was the casein GT³, a series of hybridization assays were done following theprotocol outlined in section 1.A, Salmonella extracts at 9×10⁶ cells/ml,Riboprobe at 1 μg/ml, and dA-676 capture probe at various concentrationswere used for the test. The only changes made were the following. Thepreliminary hybridization, involving only rRNA and riboprobe, was doneovernight. Control target (E. coli rRNA) was present in all reactionmixtures, not just mixtures of "heterologous cells", in order to moreclosely approximate `real world` conditions, eg conditions under whichone is looking for a specific target in a sea of background. Finally,varying amounts of capture probe were added to the reaction mixtures,resulting in final concentrations of 0-1.0 μg/ml. The results are shownin FIG. 3. Probe concentrations in the legend are in μg/ml.

From this experiment, one can see that approximately 0.25 μg/ml is theoptimal capture probe concentration. At lower concentrations, thehybridization rate and levels were not quite as advantageous. At higherconcentrations, and after a short period of rapid hybridization, theamount of captured signal actually began to drop indicating the lowereddesirability of such higher concentration of probe.

It may be noted that in the experiment presented in FIG. 3, the highestnumber of ribosomes (riboprobes) captured per cell was 35-40,000,somewhat lower than in other experiments done with this reagent. Asubsequent experiment showed that the reason for this was the presencein the reaction mixtures of heterologous cells whose rRNA couldhyper-hybridize to the riboprobes, thus removing from the mixtures aconsiderable fraction of the capturable signal. Without any heterologouscells present, the number of riboprobes captured per cell was greaterthan 100,000, as had been observed previously. This observationunderscores the importance of the protocol wherein potentiallycompetitor rRNA's are ideally removed prior to hyperhybridization (Table6).

6) Dose-Response: Campylobacter jejuni

In order to demonstrate the clinical utility of this assay, adose-response experiment was done. Various dilutions of Campylobacterjejuni extracts were probed for in a standard assay format, e.g. 10⁴-10⁶ /ml. Each assay tube contained E. coli extracts at 1.6×10⁷ cells/mlas competitors. Extracts and ³² P-labeled 5'-Riboprobe were initiallyincubated at 37° C. for 30 minutes, after which dA-tailed capture probe#732 (Campylobacter-specific) was added and the mixture incubated for anadditional 30 minutes. After this, the hybridized complexes werecaptured on magnetic beads. In one set of assays, at this point theamount of ³² p bound to the beads was determined by scintillationcounting (results below under "single capture"). In a parallel set ofassays, the complexes were eluted from the beads with 2.5M GuSCN andrecaptured on fresh beads, and the beads were counted (listed belowunder "second capture"). The data are presented in Table 7.

                  TABLE 7                                                         ______________________________________                                        Dose-Response with Campylobacter extracts                                     Target Cells                                                                           cpm       Signal to cpm     Signal to                                per sample                                                                             captured  Noise     captured                                                                              Noise                                    ______________________________________                                        1.09 × 10.sup.6                                                                  696,000   93:1      304,000 2500:1                                   1.2 × 10.sup.5                                                                   169,000   23:1      19,700  1600:1                                   1.4 × 10.sup.4                                                                   27,000    3.6:1     1,300    11:1                                    ______________________________________                                    

As can be seen from these data, the lower detection limit in this caseis less than 10⁴ cells (a Signal/Noise ratio of 3:1 was chosen as thelower limit). The elution/recapture step advantageously increased thesensitivity of this particular assay by a factor of three (3).

7) Alternative assay formats

Evidence presented in Section 2 demonstrates that the basis of GT³hyperhybridization is RNA co-precipitation. With such a nonspecificlabeling scheme, there is a potential for high background levels inassays. To obviate this difficulty, a modification is ideally made inthe assay protocol to provide an opportunity for complementary moleculesto specifically and selectively hybridize before they precipitatenon-specifically. The altered format, which is hereafter referred to asFormat II, is as follows:

a) Two microliters of target extracts (and competitor) were mixed with100 μl of GuSCN solution containing 10% Dextran Sulfate.

b) Capture probe was added (to a final concentration of 0.2 μg/ml) andthe mixture was vortexed; then the 3'-riboprobe was immediately added(final concentration 0.5μg/ml) and the mixture was again vortexed. Theresulting mixture was incubated at 37° C. for 2-3 minutes.

c) An equal volume (100 μl) of TMA.TFA/5% dextran sulfate solution(pre-warmed to 37° C.) was added, and the mixture thoroughly mixed andincubated at 37° C. for 15 minutes.

d) Prewarmed beads (200 μl) were added, and the mixture was vortexed andincubated at 37° C. for 6 minutes to effect capture.

e) The beads were separated magnetically and washed twice with 0.5MGuSCN wash buffer. (In some cases, the beads were counted to determinethe amount of cpm's captured at this first round.)

f) Targets were chemically eluted by incubating for 3 minutes at 37° C.with 100 μl of 2.5M GuSCN.

g) The eluant was separated and transferred to new tubes. New beads wereadded and the mixture was incubated for 3 minutes at 37° C.

h) The beads were separated, washed twice with 0.5M GuSCN wash bufferand counted.

The following (Table 8) are typical results from

experiments using Format II. Listeria monocytogenes or Campylobacterjejuni extracts were used as targets at a concentration of 5×10⁵cells/ml, and E. coli was present at 2.6×10⁷ cells/ml as a competitor. Acontrol reaction contained E. coli but no true target extracts. Captureprobes used in these experiments were: dA-795 capture probe for Listeriaand dA-732 for Campylobacter.

                  TABLE 8                                                         ______________________________________                                        Assay performance using "Format II"                                                    Capture Signal  Riboprobes                                                                            E. coli                                      Sample   Round   cpm     per cell                                                                              cpm   S/N                                    ______________________________________                                        Listeria 1       372,000 747,000 3,400 220:1                                           2       302,000 605,000 130   2,400:1                                Campylobacter                                                                          1       422,000 847,000 51,000                                                                               8:1                                            2       211,000 423,000 2,700  77:1                                  ______________________________________                                    

As with other formats, assays with both Listeria and Campylobacterdemonstrated "hyperhybridization" by their exceedingly high riboprobe tocell number. E. coli gave fairly high backgrounds with probe 732 (seeU.S. Ser. No. 216,679 for further details) after the first capture roundin this assay, but most of the noise was eliminated by means of achemical elution followed by a second capture.

The same format was tested with Neisseria gonorrhoeae extracts (5×10⁵cells/ml) while extracts from the close relative, Neisseriameningitidis, served as the control. In this experiment, the riboprobeconcentration was lowered to 0.2μg/ml final concentration but still theformat resulted in the capture of an extremely high number ofriboprobes/cell, indicating "hyperhybridization".

The following formats address the issue of increasing the assayspecificity with reagents such as GT³, which can otherwise causenonspecific labeling. One potential problem with a nonspecific labelingscheme is that the specificity of the assay depends solely on thespecificity of the single oligonucleotide capture probe. In many casesthis does indeed provide sufficient specificity. For example, in theabove Neisseria Format II experiment, hyperhybridization is sufficientlyspecific to distinguish N. gonorrhoeae from its closest relative, N.meningitidis. The 28-mer capture probe employed in that experiment hadonly 4 nucleotide mismatches with N. moningitidis, of which 2 were G:Tbase-pairs. In essence, then, there were effectively only two strongmismatches; yet the assay readily distinguished them. This was due tothe highly specific hybridization obtained in GT³ of the N.gonorrhoeae-specific oligonucleotide to its target in GT³. Data willalso be presented below (Tables 10 and 11) demonstrating that 10² N.gonorrhoeae cells were detected using hybridization with only onespecific oligonucleotide in GT³ in the presence of a 10,000- or100,000-fold excess of N. meningitidis.

One advantage of sandwich-type assays such as reversible target captureis that the specificity is determined by the specificity of at least twoprobes. In some cases, such specificity may be necessary. One skilled inthe art will realize that the following format, to be termed Format III,provides methods whereby such a highly desired level of specificity canbe achieved.

Format III

a) Hybridize a first specific oligonucleotide capture probe to a targetin a suitable (nonprecipitating) reagent.

b) Capture on a first solid support.

c) Elute and rehybridize a second specific oligonucleotide capture probe(containing a second mixed-base sequence distinct from the mixed basesequence of the first probe) to the target.

d) Capture the target:probe complex on a second solid support via thesecond specific capture probe.

e) Elute the target:probe complex from the second solid support.

f) Add a suitable labeled probe and the reagents necessary to effectsuperstoichiometric labeling.

g) Use further cycles of release/recapture to reduce any nonspecificbackgrounds as necessary.

h) Detect labeled targets

The following comments apply to this format:

a) The repetition of capture/release cycles lends itself most naturallyto an automated assay.

b) The most preferred reagents of the present invention forhybridization to the target would be those possessing as many aspossible of the desirable properties listed in the Summary of theInvention (above), except the ability to effect superstoichiometriclabeling. (Precipitation of heterologous RNA and other cellularcomponents would not be desirable in steps 1-4.)

c) The capture probes and the solid supports would preferably possesscomplementary members of affinity pairs. The affinity pairs employed inthe first and second captures can be the same or similar in nature, ordifferent in composition.

The following are examples of uses of a Format III assay.

1) Using the same or similar affinity pairs for the two captures

Both capture probes are ideally dA-tailed. The hybrid between themixed-base sequence of the first capture probe and the target isadvantageously less stable than that between the dA-tail and theoligo-dT or poly-dT on the support. Thus, elution of the target from thesupport can be carried out without separating the first capture probefrom the solid support. In addition, the mixed-base sequence of thesecond capture probe binds to the target to form a hybrid which is morestable than the hybrid between the dA-tail of this probe and theoligo-dT on the support. This enables the target to be repeatedlyrecaptured via the second oligonucleotide probe for reduction ofnonspecific background.

One skilled in the art will realize that substantial differences in thelengths of the relevant mixed-base sequence and homopolymer sequencescan be exploited to accomplish this. For example, one could use a shortfirst mixed-base sequence and a long poly-dT on the first solid support,and a long mixed-base sequence on the second capture probe and a shortoligo-dT on the second solid support.

The present invention provides additional, useful, and novel methods:tetraalklyammonium salts preferentially stabilize poly-dA:poly-dTbase-pair bonds. Poly-dA:poly-dT, for example, has a T_(d) of about 65°C. in 2.4M TEA.Cl, while a 35-mer mixed-base sequence has a T_(d) ofapproximately 43°-44° C., regardless of GC content (Woods et al. (1985)Proc. Natl. Acad. Sci. U.S.A., 82, 1585-1588). In the present example,this preferential strengthening of poly-dA:oligo-dT or poly-dA:poly-dTby tetraalkylammonium salts can be exploited to enhance the retention ofthe first capture probe on the first solid support during the elution ofthe target.

It is worth noting that this preferential stabilization ofpoly-dT:poly-dA by tetraalkylammonium cations can also be readilyexploited in a useful generic stringency wash procedure with a targetimmobilized via poly-dT:poly-dA bonds. For a fixed oligonucleotide probelength, mismatched mixed-base sequences (pseudo-targets) can readily beremoved from the solid support in a manner independent of GC content(i.e., with a single set of wash conditions) without any loss of genuinetarget from the support; this is accomplished by incubation just belowthe T_(d) of the true target-probe hybrid.

2) Using different affinity pairs for the two captures

The first capture probe can be derivitized with biotin and the firstsolid support derivitized with streptavidin. The second capture probe isthen advantageously dA-tailed and the second solid support coated witholigo-dT. In this case, both capture probes could be added in the firststep of the assay, and the first elution could remove the target with orwithout the first capture probe.

One skilled in the art will readily realize that the affinity pairscould also be reversed: poly-dA:oligo-dT for the first capture andbiotin:streptavidin for the second. (Additional dA-tailed capture probecan be added after the second elution if necessary.) The reversibilityof the poly-dA:oligo-dT interaction could then be used to reduce thenonspecific binding of labeled probe to the support in subsequent steps.

One skilled in the art will further realize that the above methods andthe reagents of the instant invention can be advantageously combined innumerous ways to gain even more specificity (for example, using three ormore oligonucleotides). Furthermore, other affinity pairs such asantibody:antigen pairs, and/or any other pairs, may confer even greaterspecificity in the practice of the present invention, which employs anonspecific labeling reaction with a generic labeled probe in thepreferred method. Indeed such other methods of derivitizing andproviding for specific ligand-ligand binding partner interactions becomeself-evident.

8) Dose-Response:Neisseria gonorrhoeae

The sensitivity of the assay (Format II) was examined using N.gonorrhoeae at 10⁵ to 10² cells/ml; N. meningitidis was present in allassays at 5×10⁵ cells/ml.

After a second capture, N. gonorrhoeae could be clearly seen abovebackground at as low as 10² cells/ml. (The S/N ratio was 3/1 or 7/1, forNeisseria meningitidis and E. coli control organism, respectively.) Thelevel of hyperhybridization observed with 10² cells/ml was almost 8million probes per cell, which corresponds to nearly 800 probes pertarget.

A repeat experiment was performed using only 0.05 μg/ml riboprobe andthe N. meningitidis competitor was increased to 10⁷ cells/ml. Targetswere captured/recaptured three times.

Despite using only 1/4 the previous amount of riboprobe, N. gonorrhoeaeat 10² cells/ml still could be seen above elevated competitor levels(S/N approximately 6:1).

The signals at 10² N. gonorrhoeae/ml were comparable to those at 10³/ml, and both were well above backgrounds. While not wishing to be boundthereby, the explanation for this would seem to be that there are farmore riboprobes captured per cell as the ratio of added labeled probesto target molecules increases. We have discovered that this unexpectedphenomenon is useful since the need for greater signals is most criticalat low target concentrations. Hyperhybridization with the instantreagent thus promotes this enhancement precisely where it is mostneeded.

9) Dextran Sulfate not necessary for hyperhybridization

Since "hyperhybridization" appears to be a precipitation phenomenon, theassays were performed in the presence of 0%, 2%, 4%, 6%, 8% and 10%(mass/volume) dextran sulfate (A stock (65%, mass/volume) solution wasprepared by, e.g., dissolving 65 g Dextron Sulfate in buffered water,and adjusting the final volume to 100 mL: the result was a "65%"solution. An appropriate volume of this stock was added to each reactionmixture to give a final concentration as desired {2%-10%}.) usingListeria as a model to determine whether dextran sulfate was necessarywith GT³. These experiments showed that dextran sulfate had noappreciable affect on the signal. The average signal was approximately250,000-300,000 riboprobes/cell with an average E. coli S/N of 500:1after a second capture (the target cell concentration was 10⁵ cells/ml).

10) Concentration of Riboprobe

An experiment was done to compare how much riboprobe could be used toadequately detect a number of target levels in GT³ versus GuSCN. Again,N. gonorrhoeae was used at 10⁵ and 10⁴ cells/ml, with 0.2 μg/ml captureprobe, and riboprobe concentrations of 0.2 μg/ml, 0.05 μg/ml and 0.0125μg/ml. N. mengitidis was present as a competitor at 5×10⁵ cells perassay. Two rounds of capture were performed. The results indicated thatas little as 12.5 μg/ml riboprobe was sufficient to detect target atwell above competitor background. This was some forty-fold less than theconcentration required for efficient labeling in GuSCN, where themechanism of labeling is classical hybridization. A considerable savingsin assay cost is thus an added and unexpected advantage ofhyperhybridization in GT³.

F. Reporter probes:Riboprobes vs. oligonucleotides

It was further discovered that the observed "hyperhybridization"phenomenon in GT³ generally correlates with the presence of a Riboprobeof either the 3'- or 5'-variety. This is useful to recognize since insome instances, in order to increase the selectivity of an assay, it maybe desirable to use a target-specific reporter-oligomer (in combinationwith a specific capture probe) rather than a generic rRNA Riboprobe. Insuch instances since superstoichiometric labeling generally occurs onlyif the labeled probe is a Riboprobe, the preferred way to increaseselectivity is to develop an assay format using two specific captureprobes and subsequently a nonspecific labeling reaction with aRiboprobe.

G. RNA:DNA Hybrid melting

A nucleic acid melting curve reveals important information about areagent and the hybrids formed therein. Specifically, it indicateswhether a true hybrid is present (if so, it will have a clear meltingpoint) and whether or not GC and AT base-pairs are equally strong ifthey are, the melting curve will be sharp, and the melting/dissociationtemperatures (T_(d) 's) of various hybrids of the same length butdifferent GC/AT content will be nearly equal. GT³ melting experimentswere conducted with a variety of target rRNA's, reporter probes andcapture probes, as listed in the following table.

                  TABLE 9                                                         ______________________________________                                        Target/probe combinations used in melting studies                                             Labeled  Tailed                                                               (reporter)                                                                             (capture)                                            Target rRNA     probe    probe                                                ______________________________________                                        Campylobacter   Riboprobe                                                                              dA-732                                               E. coli         Riboprobe                                                                              dA-787                                               Salmonella      Riboprobe                                                                              dA-676                                               ______________________________________                                    

Hybrids were formed in GT³ by overnight incubation at 37° C. with targetcell extracts at 10⁷ -10⁸ cells/ml, reporter probe at 1 μg/ml andcapture probe at 0.25 μg/ml. This hybridization mixture was then dilutedby 1/10 with GT³ and held at 37° C. for the course of the meltingexperiment.

Aliquots were removed periodically and incubated at various elevatedtemperatures for six minutes, after which the intact complexes werecaptured on magnetic beads, washed and counted as described in section1.A. As a control, periodically over the course of the experimentaliquots held at 37° C. were subjected to the capture procedure tomonitor any changes in the level of hybridization over time. The numberof intact (i.e., captured) hybrids at each elevated temperature,relative to the number of intact hybrids in the 37° C. control tube, wascalculated.

                  TABLE 10                                                        ______________________________________                                        Melting of various target/probe complexes                                     using a .sup.32 P-Riboprobe as reporter (from FIG. 6)                                  Capture  GC content         Width*                                   Target rRNA                                                                            Probe    of probe   T.sub.d (ΔT3/4-1/4)                        ______________________________________                                        Campylobacter                                                                          dA-732   46%        51-52° C.                                                                      ≦5° C.                     E. coli  dA-787   37%        48° C.                                                                         ≦5° C.                     Salmonella                                                                             dA-676   43%        49° C.                                                                         ≦7° C.                     ______________________________________                                         *Curve width is, by standard definition, the temperature range over which     one observes a transition from 3/4 of the hybrids being intact to 1/4         being intact. The actual widths are suspected to be equal to or less than     those reported in this table because the temperaturepoints used in the        experiment were 5° C. apart: by sampling more frequently one can       generally obtain sharper curves.                                         

As can be seen, the T_(d) 's are within 3°-4° C. of each other and themelting curves have widths (ΔT_(3/4-1/4)) of 5°-7° C. These widths arecomparable in size to the spacing of the data points (5° C.). Theseresults can be compared to our results obtained in 3.0M TMACl where theΔT_(3/4-1/4) was 6°-8° C. TMACl is a reported GC/AT equalizer (Melchiorand von Hippel, PNAS 70 (1973), 298-302). The similarity of thedissociation temperatures indicates that the desired effect of GC/ATequivalence was obtained.

H. DNA:DNA melting in GuSCN:TMA.TFA combination reagents

In section 1.G the data associated with DNA:RNA melting in GT³ weresharp indicating good specificity of base-pairing and a reasonabledegree of GC/AT equivalence. This can also be demonstrated with aDNA:DNA oligomer:oligomer system (one oligomer radio-labeled, the otherone dA-tailed for bead-capture) and because of its greater simplicity(compared to the system containing a reporter probe (labeled Riboprobeor oligomer), a capture probe, a target RNA and the assorted componentsfrom cell extracts), such a system is more easily interpreted.Additionally, using oligomers, one can choose almost any sequence, witha wide range of GC-content, for the experiments. In this section,melting experiments were carried out with such a DNA:DNA system asdescribed.

The basic protocol for these experiments was the same as that describedin section 1.G. In GT³, hybridization was accomplished by incubation at37° C. Hybridization in GuSCN was advantageously carried out at roomtemperature, as the relatively low hybrid melting temperature was foundto contraindicate 37° C. incubation. In the hybridization mixture, the³² P-labeled oligomer was present at 50 μg/ml, and the dA-tailed captureprobe at 100 μg/ml. Hybridization was found to occur rather rapidly(reaching a plateau within 5 minutes in one case), and the hybrids werestable at the incubation temperature for at least two days; hybridsformed in this way could be used in melting experiments after a shortincubation or after a day or two of incubation. Initially, thehybridization mixture was diluted 10-fold prior to carrying out meltingexperiments; after the protocol, a 40-fold dilution was discovered to beoptimal.

Two pairs of oligomers were used for the melting experiments. Of eachpair, one oligomer (the target) was 5'-³² P-labeled and the other (thecapture probe) was dA-tailed. The sequences of the target probes were E.coli rRNA sequences, and the capture probe sequences were complementarythereto.

All oligomers were 35 nucleotides in length. The two pairs were chosento represent sequences of minimal and maximal GC-content. The oligomerschosen were as follows:

    ______________________________________                                        Target oligomer                                                                              Capture probe                                                                            GC-content                                          ______________________________________                                        #855           dA.sub.112 -730                                                                          37%                                                 #853           dA.sub.125 -854                                                                          66%                                                 ______________________________________                                    

The following dissociation temperatures (T_(d) 's) were calculated fromthe melting curves obtained with these two pairs of oligomers in 2.5MGuSCN and GT³ :

                  TABLE 11                                                        ______________________________________                                        Dissociation Temperatures of DNA:DNA Hybrids                                  GC-content      in GuSCN in GT.sup.3                                          ______________________________________                                        37%             39° C.                                                                          49° C.                                        66%             54° C.                                                                          57° C.                                        ______________________________________                                    

These data indicate that GT³ is nearly twice as effective aGC/AT-equalizing reagent than is GuSCN: in the former the difference inthe T_(d) 's of the two pairs of oligomers was 8° C., while in thelatter it was 15° C. It was also discovered that in GT³ the DNA:DNAdissociation temperatures were quite similar to DNA:RNA T_(d) 's whichunexpectedly makes the reagent especially useful for the standardizationof instrument-based assays, particularly if such have the capability ofdoing either DNA:DNA or DNA:RNA hybridization assays. The low T_(d) (39°C.) of a 37%-GC 35-base-pair DNA:DNA hybrid in GuSCN should also benoted: this indicated that there would be a problem hybridizing low-%-GCsequences of 35 or fewer nucleotides at 37° C. in GuSCN, and thusfurther points to the utility and advantage of the present reagents inequalizing the GC/AT base-pairs.

A 40:60 mixture of 5M GuSCN and ⁻ 4M TMA.TFA was tried to further reducethe difference in the T_(d) 's. (In the present document this mixturewill be referred to as 40:60 GT³, or simply as GT³ *; it is anothermember of the GT³ family.) Melting experiments were carried out in thisreagent (protocol as above), and from the results the T_(d) 's werecalculated and are listed below.

                  TABLE 12                                                        ______________________________________                                        Dissociation Temperatures of DNA:DNA Hybrids                                  GC-content      in GuSCN in GT.sup.3 *                                        ______________________________________                                        37%             39° C.                                                                          52° C.                                        66%             53° C.                                                                          58° C.                                        ______________________________________                                    

As these numbers indicate, 40:60 GT³ is a still more preferred GC/ATequalizer than 50:50 GT³. The difference in T_(d) 's of the two oligomerpairs is 6° C. in the former, compared to 8° C. in the latter. Acomparison of the data revealed that the decrease in the difference wasapparently due primarily to the strengthening of the AT base-pairs,since the T_(d) of the AT-rich oligomers changed more than that of theGC-rich oligomers.

The following experiments tested the 40:60 GT³ 's ability to promotehybridization and inhibit RNase.

I. 40:60 GT³ :Signal and Noise, and RNase inhibition

Using Campylobacter extracts as a target, and E. coli extracts as acontrol, the level of hybridization in 40:60 GT³ was shown to be3-5-fold higher than in 50:50 GT³. The noise level increased by the sameamount, so the Signal/Noise ratio remained constant thereby indicatingno further hybridization enhancement.

Next, an RNase A inhibition experiment was carried out as described insection 1.D. Pre-formed hybrids were diluted into 0.1M Phosphate (pH7.55), 2.5M GuSCN, GT³ (50:50) or GT³ * (40:60). After incubation in thepresence of various concentrations of RNase A, intact targets werecaptured on magnetic beads and the fraction of intact targets (relativeto a no-RNase control) was determined. The results are presented in FIG.6. In this figure, the fraction of intact targets, which is proportionalto the level of RNase inhibition, is presented as a function of RNase Aconcentration. Clearly GT³ * and GT³ effectively inhibit RNase A. Thus,decreasing the fraction of GuSCN in the combination reagent from 50% to40% advantageously provided no adverse effect in terms of nucleaseprotection.

J. Cellular lysis

A series of experiments was performed to assess the ability of the newreagent to lyse bacterial cells. The lysis capabilities of the newreagents were compared to those of SDS/Proteinase K and/or GuanidiniumThiocyanate, both of which had been shown to be useful as lysing agents.The standard method for determining the extent of lysis is to determinethe relative decrease in the optical density (O.D.) at 550 nm of analiquot of cells after a 15 minute incubation in the medium of interestat a specified temperature. We used E. coli and four Salmonella species(brookfield, bulawayo, minneapolis, typhimurium) as the target cells inour studies.

As noted earlier, in a typical experiment SDS (0.5%) plus Proteinase K(0.5-1.0 mg/ml) lysed (5-10 minutes at 55°-65° C.) 90-100% of an aliquotof cells. GuSCN (2.5M) plus Sarkosyl (0.5%) typically lysed (15 minutesat 37° C.) 75-95% of the target bacterial cells; the addition ofProteinase K to this lysis cocktail was found not to increase the extentof lysis.

Lysis experiments were carried out with GT³ (a 1:1 mixture of 5M GuSCN(buffered with Tris/EDTA) and 4M TMA.TFA (buffered with 100 mMphosphate, 100 mM borate and 50mM EDTA)) and GT⁵ (a 1:1:1 combination of5M GuSCN, 4M TMA.TFA and 4M TMA.SCN (buffered as TMA.TFA was)) andvarious combinations of additives: DTT (0.5%), Sarkosyl (0.5%),Proteinase K (0.5 mg/ml). Incubation was at 37° C. or 65° C. The dataobtained with each tested combination of additives and the fivebacterial species mentioned above were averaged. Since there was nosignificant difference in lysis as a function of temperature the dataobtained at both temperatures were analyzed together. The results aresummarized in Table 13.

                  TABLE 13                                                        ______________________________________                                        Extent of cellular lysis in GT.sup.3 and GT.sup.5                                          Extent of Lysis                                                  Additive       GT.sup.3 GT.sup.5                                              ______________________________________                                        none           47% ± 2%                                                                            43% ± 3%                                           Sarkosyl       66% ± 3%                                                                            63% ± 2%                                           Sark., DTT     78% ± 4%                                                                            73% ± 5%                                           Sark., PK      85% ± 3%                                                                            89% ± 3%                                           ______________________________________                                    

Under "Extent of Lysis," the uncertainties (±) are Standard Errors ofthe data.

From these data, it became apparent that the lysis efficiency of eitherGT³ or GT⁵ could be improved by the addition of any combination of theadditives listed with the Sarkosyl/Proteinase K combination being mostpreferred.

An alternative approach to the development of an assay with GT³ as thehybridization medium, is to first lyse the target cells with GuSCN, SDSor Proteinase K, which will result in a very good overall detectionefficiency.

K. Assay performance with a biological specimen

The experiments reported above all involved the detection of targetsfrom bacterial cell extracts. To evaluate the performance of GT³ undermore `real` conditions, an experiment to detect targets in nonfat drymilk (NFDM) was performed. The protocol was as follows.

NFDM was dissolved in 5M GuSCN (plus 1% Sarkosyl and 10% by weightdextran sulfate) to a final concentration of 10% (w/v). One volume ofthis solution was then combined with one volume of 4M TMA.TFA (plus 7%by weight dextran sulfate). The resulting solution was "GT³ " with 5%NFDM, 0.5% Sarkosyl and 8.5% dextran sulfate: hereafter referred to as"NFDM/GT³." To this mixture Listeria extracts (experimental target) wereadded to a final concentration of 5×10⁵ cells/ml, or E. coli extracts(negative control) to a final concentration of 10⁸ cells/ml. As apositive control, Listeria extracts were added to GT³ (with dextransulfate and Sarkosyl) to a final concentration of 5×10⁵ cells/ml. AListeria-specific capture probe (dA-tailed #795) was added to allsamples at 0.2 μg/ml, and a ³² P-labeled 3'-Riboprobe at 0.5 μg/ml.After a 15-minute incubation at 37° C., one volume of magnetic beads wasadded and the samples were incubated at 37° C. for 6 minutes. Beads werewashed three times, after which the samples were eluted in 2.5M GuSCNand then recaptured by the addition of two volumes of beads. The beadswere washed twice and counted. The results were as follows:

                  TABLE 14                                                        ______________________________________                                        Hybridization in Nonfat Dry Milk in GT.sup.3                                  Listeria     Listeria in                                                      in GT.sup.3  NFDM/GT.sup.3 E. coli in                                         R'probes     R'probes          NFDM/GT.sup.3                                  per cell     per cell cpm (S)  cpm (N)                                                                              S/N                                     ______________________________________                                        Bead-bound,                                                                           541,000  258,000  311,000                                                                              14,100   22:1                                first round                                                                   Eluted  448,000  224,000  258,000                                                                              6,600    39:1                                Bead-bound,                                                                           384,000  143,000  221,000                                                                              23     9,600:1                               second round                                                                  ______________________________________                                    

From these data it was apparent that:

1) The differences in labeled probes captured per cell in GT³ andNFDM/GT³ indicate that there was some competition for the labeled probesin NFDM. (In a similar experiment where the Riboprobe was bound totargets only after a preliminary round of target capture to removecompetitors, there was no signal reduction due to competitors in NFDM.)

2) Even in the presence of NFDM, the extent of hyperhybridization wasstill very great: after two rounds of capture, 143,000 probes were boundper cell (compared to 384,000 without NFDM).

3) The background levels were low after two rounds of capture: in thepresence of NFDM, the S/N ratio was nearly 10,000:1 (with only 10⁵ cellsin the sample). This sensitivity is far more than adequate to detectcontaminating bacteria in foods since a typical food sample contaminatedwith only one Listeria cell gives rise to at least 10⁶ cells/ml afterstandard (BAM/AOAC) cultural enrichment.

4) Additional assay steps, such as a final filter capture, mayadvantageously enhance the sensitivity even further.

L. A GT³ Elution Buffer

The designation "GT³ " refers not to a single reagent but to a family ofreagents containing GuSCN, TMA.TFA and buffers, combined in variousratios. When the combination ratio is not specified, it is understood tobe 1:1, referring to the medium in which most of the experimentsdescribed herein were done. However, variations on this basic reagenthave been prepared and have been shown to have properties similar tothose of the basic reagent. Examples of such preferred variants are:40:60 GT³ (GT³ *) and GT⁵. The former of these was more preferred sinceit conferred a greater degree of GC/AT equivalence on hybrids than didthe 1:1 reagent.

One variant which has been shown to have desirable properties for aspecial application is an elution buffer comprising mostly GuSCN (themain component of the standard chemical elution buffer), with a smallamount of TMA.TFA added. To evaluate this buffer, the followingexperiment was carried out. In GT³, hybrids were formed involving a ³²P-labeled Riboprobe, a dA-tailed capture probe, and rRNA from extracts(at 5×10⁵ cells/ml) of Salmonella typhimurium and Neisseria gonorrhoeae.With the Salmonella capture probe a control experiment was run withextracts from E. coli (5×10⁷ /ml), while with the N. gonorrhoeae probe acontrol was run with N. meningitidis extracts. After hybridization, themacromolecular complexes were captured on magnetic beads, and the beadswere washed once with 0.5M GuSCN Wash Buffer (at room temperature), oncewith 2.0M GuSCN Wash Buffer (for 2 minutes at 37° C.), and one finaltime with 0.5M GuSCN Wash Buffer (room temperature). Elution buffer (ofvarious compositions, as listed below; prewarmed to 37° C.) was thenadded to the beads and incubated at 37° C. for 2 minutes, after whichthe eluate was transferred to a clean tube. The Elution Buffers usedwere as indicated in the following table.

    ______________________________________                                        Designation     GuSCN   TMA.TFA                                               ______________________________________                                        A               3.3M    --                                                    B               3.1M    0.2M                                                  C               3.0M    0.4M                                                  D               2.6M    0.8M                                                  ______________________________________                                    

The eluted complexes were then recaptured on magnetic beads, and thebeads were washed three times with 0.5M GUSCN Wash Buffer (at roomtemperature) and counted in a scintillation counter.

The results of this series of experiments are presented in Table 15.

                  TABLE 15                                                        ______________________________________                                        Elution in GuSCN and "GT.sup.3 -type" buffers                                           Signal (cpm) Control (cpm)                                                  Elution 1st      2nd     1st    2nd                                   Target  Buffer  capture  capture capture                                                                              capture                               ______________________________________                                        Neisseria                                                                             A       318,000  13,100  8,300  690                                   gonorrhoeae                                                                           B       321,000  88,700  7,700  140                                           C       338,000  133,000 7,100  420                                           D       327,000  61,000  6,600  680                                   Salmonella                                                                            A       223,000   2,920  3,100   34                                           B       218,000  21,700  6,300  150                                           C       229,000  63,400  2,400   84                                           D       248,000  89,900  4,200  170                                   ______________________________________                                    

From these data it can be seen that the addition of a small amount ofTMA.TFA (to a final concentration of 0.2-0.4M) to the elution bufferadvantageously enhances the post-elution recapture in both cases, thoughto different extents depending on the target. Background levels did notincrease to the same extent as signals thereby increasing the S/Nlevels.

It was thus discovered that a GuSCN elution buffer with a small amountof TMA.TFA has the capability of advantageously improving assayperformance by improving post-elution recapture on magnetic beads. Ahypothetical explanation for this is that the TMA.TFA, even at lowconcentrations, prevents the hyperhybrids (precipitates: see below) fromdisaggregating, thus minimizing signal loss through theelution/recapture process. Those skilled in the art will now recognizethat by optimizing the precise composition of the elution buffer one canmaximize both signal preservation and noise reduction.

II. Mechanism

In the course of experiments with the new reagents of the presentinvention, the observation was made that a substantial precipitationoccurred upon the addition of 4M TMA.TFA to concentrated (>10¹⁰ E.coli/ml) cell extracts in 5M GuSCN. The precipitate formed was largeenough to settle out of solution under the force of gravity. Since GT³is a 1:1 mixture of 4M TMA TFA and 5M GuSCN, the nature of thisprecipitate and its possible role in hyperhybridization in GT³ wasquestioned. The formation in GT³ of a precipitate, comprised of thetarget molecule as well as the hybridized capture probe and a largenumber of labeled riboprobes, would be a possible explanation for thephenomenon of hyperhybridization, in which a very high number ofriboprobes are detected per target molecule. If precipitation does playa role in hyperhybridization, it is important to fully understand itsnature in order to control it, to enhance it, and in certain cases tomake it selective. It was surprisingly discovered that indeed, with thereagents of the present invention, the selectivity of the precipitationcan be controlled, thereby allowing for the development of a very rapidRNA isolation procedure.

A number of experiments were performed to define the nature of theprecipitate including measuring the amount and type (eg dsRNA, ssRNA,cDNA, etc) of nucleic acids which precipitated in GT³, nucleic acidprecipitation at various temperatures and at various cellconcentrations, and whether the precipitate could be dissolved andre-precipitated. It was surprisingly discovered that little differencewas seen in the precipitation of the riboprobe (ssRNA) on ice, at roomtemperature, at 37° C., or at 65° C., indicating that hyperhybrids willprobably be stable even at 65° C. in GT³. The precipitation of the dsDNAwas fairly constant at the three lower temperatures, but did show asharp decrease at 65° C. The differential stability of dsDNA and theriboprobe would ideally be exploited as follows: backgrounds is(resulting from the presence of dsDNA) are ideally removed from truehybrids (containing riboprobes) by carrying out a GT³ stringency wash ata temperature a few degrees below the hybrids' T_(d) in GT³ (53°-58°C.).

It was also discovered that oligomers do not precipitate at the samelevels as riboprobes. This demonstrates why riboprobes are preferred asreporter probes (as opposed to oligonucleotides) in order toadvantageously obtain hyperhybridization.

The precipitation of dsDNA appeared to be efficient only at very highcell concentrations and surprisingly was inhibited by the presence ofdextran sulfate. This distinction can thus be advantageously exploitedin an RNA isolation procedure, and/or a nucleic acid probe assay whereinthe target is RNA.

The experiments also demonstrated that the absolute amount of nucleicacid precipitated by GT³ varied somewhat from experiment to experimentcorrelating with the variability of the amount of hyperhybridizationfrom experiment to experiment.

A. Dissolution and re-precipitation of GT³ precipitates

In order to fully detect hyperhybridization non-isotopically in ageneric assay format, it may be necessary to dissolve the "hyperhybrid"prior to the detection step of the assay. Experiments to further explorethe hyperhybridization phenomenon demonstrated that 2.4M TEA.Cl doesindeed dissolve hyperhybridization complexes. The GT³ precipitationassay described above was used to examine the resuspension ofprecipitated nucleic acids, using 2.4M TEA Cl, thermal elution buffer(containing 0.5M NaCl), and 5M GuSCN. Results showed that bothprecipitated ssRNA and dsDNA were efficiently resuspended in all threesolutions. Resuspension in 5M GuSCN has the advantage of making possiblethe subsequent re-precipitation of the nucleic acid by the addition of4M TMA.TFA. This would be of special advantage in gaining additionalpurity in an isolation procedure. The phenomenon can also be used in anucleic acid probe assay to lessen the trapping of oligomeric captureprobes in precipitates and indeed, hyperhybridization complexes can bedissolved with thermal elution buffer and with 5M GuSCN, as well as with2.4M TEA.Cl. It has also been discovered that 2.4M TEA.Cl disrupted"hyperhybrids" formed in GT³. Virtually all signal was lost as a resultof TEA.Cl-washing. Control experiments showed that theoligonucleotide-target bond and the capture probe-bead bonds were stableto TEA.Cl-washing. Accordingly, once dissolved with 5M GuSCN, suchcomplexes can be reformed upon the addition of 4M TMA.TFA.

B. Hyperhybridization with a ss-DNA target

One also can advantageously apply hyperhybridization to ss-DNA targets,as in the following scheme. First, one hybridizes a target to a specificoligonucleotide probe; the probe also has a tail which is complementaryto a subsequently added, labeled riboprobe. Next, one adds the labeledcomplementary riboprobe. This converts the ss-DNA target, which is notprecipitable, to a complex containing ss-DNA linked to a large ss-RNAthrough the oligonucleotide probe. This ss-DNA:ss-RNA complex issignificantly different from a DNA:RNA hybrid (which does notprecipitate as well in GT³), and thus it precipitates in GT³ by virtueof its large single-stranded RNA moiety. The advantages of this methodare that (1) it makes it possible to effect superstoichiometric labelingwith ss-DNA targets, and (2) it makes it possible to use generic labeledriboprobes with non-complementary target sequences.

C. Summary: mechanism of hyperhybridization

Single-stranded (ss) and double-stranded (ds) RNA were shown toprecipitate well in GT³, while oligonucleotides, ssDNA and RNA:DNAhybrids remained soluble. Double-stranded DNA precipitated well only athigh cell concentrations (suggesting that it was merely beingnon-specifically trapped), and its precipitation was inhibited by hightemperatures and dextran sulfate. These experiments indicated thatprecipitation of ssRNA can explain hyperhybridization. For example,oligomers are soluble in GT³, while riboprobes are not. This explainswhy hyperhybridization was observed with riboprobes but not witholigomers. It also accounts for the specificity of thehyperhybridization phenomenon: the only way a tailed oligonucleotidebecomes associated with the precipitated RNA-target:RNA-probe complex isvia hybridization (except for a small amount of non-specific trapping ofthe oligonucleotide in the precipitates. This trapping is ideallyreduced by cycles of solubilizing and precipitating the RNA, or by theuse of two or more cycles of capture/release with two or more specificoligomeric capture probes.) While precipitation may occur in the absenceof hybridization of the capture probe, such background precipitates arenot capturable.

Other pertinent correlations that were discovered include the following:TEA.Cl-washes disrupted hyperhybrids and also dissolved precipitatedriboprobes, hyperhybrids were stable beyond 50° C. and the precipitatedriboprobe was stable at 65° C. in E. coli extracts processed in GT³.

Superstoichiometric labeling is preferably carried out in solutionbecause of the preferred rapid kinetics of solution-phase reactions.However, those skilled in the art will realize that in principle theprocess should also occur with the target immobilized on a solid support(either directly, or indirectly through a capture probe). One skilled inthe art will further realize that the kinetics of such a process may notbe compatible with rapid diagnostic assays, but may be enhanced by theaddition of an appropriate accelerator(s) such as, but not limited to,dextran sulfate.

Contrary to Kohne's explanation of accelerated hybridization (seeBackground) the uniqueness and significance of the novel reagents of theinvention is that not only has the rate of hybridization been greatlyenhanced, but so also has the magnitude of the hybridization signal,without a proportional increase in hybridization background.

Comparison of the Conventional Assays with the Preferred Methods usingGT³

An experiment was done to compare conventional hybridization in SET("1×SET" consists of 150 mM NaCl, 1 mM EDTA, 10 mM Tris HCl, pH8.0)buffer on nitrocellulose filter membranes with the preferred embodimentof the present invention. Listeria monocytogenes cell extracts werediluted to various concentrations, spotted on nitrocellulose filterswith a diluent-only spot as a control, and affixed by baking themembranes. All filters were prepared in duplicate. Hybridizations werethen carried out following standard procedures (overnight incubation at65°-68° C.), using a 32p-labeled Riboprobe as the reporter probe. Thefilters were then rinsed (as per standard protocols) and subjected towashes of various stringency levels (1.5x, 1.0x and 0.5x SET). Afterdrying, the spots were cut out, dried and counted. Signal-to-noiseratios were calculated (the diluent-only spot was the "Noise"). The 0.5xSET stringency wash gave rise to the best S/N ratios, and data fromthese filters are presented in the following table:

    ______________________________________                                        Filter Hybridizations in SET:                                                 Listeria Dose-Response                                                        Cells per spot                                                                              Signal/Noise Ratio                                              ______________________________________                                        1.5 × 10.sup.6                                                                        136                                                             4.7 × 10.sup.5                                                                        30                                                              1.5 × 10.sup.5                                                                        8.3                                                             4.7 × 10.sup.4                                                                        1.5                                                             1.5 × 10.sup.4                                                                        0.6                                                             ______________________________________                                    

Since a Signal/Noise ratio of 3 is ordinarily taken as the cut-off fordetection sensitivity, these data indicate a sensitivity of 1.5×10⁵cells.

A similar dose-response experiment was carried out using the preferredliquid-phase hybridization method in GT³, followed by three rounds ofreversible target capture, as previously described. The results were asfollows:

The final ion concentrations were as listed below

    ______________________________________                                        Reagent  TMA     Gu          SCN   TFA                                        ______________________________________                                        A        5.0M    --          5.0M  --                                         B        4.0M    --          2.5M  1.6M                                       C        2.5M    2.1M        4.5M  --                                         ______________________________________                                    

The hybridization in TMA.SCN/GuSCN (Reagent "C") was generally much lesspreferred. Preferred melting curves were obtained with the otherreagents thereby demonstrating that the mixed salt (reagent "B",hereafter referred to as "TMA.TFA/SCN" or "T³ ") conferred meltingcharacteristics at least as advantageous as those observed in TMA.SCNalone.

Similar RNA:DNA melting experiments were conducted with T³ following thegeneral experimental protocol outlined in section 1.A. The final saltconcentration was ≈3.6M TMA, ≈1.3M TFA, ≈2.1M SCN. The resultant meltingcurve was clearly much sharper (^(T) _(3/4-1/4) =≈9° C.) than wasobtained in TMA.TFA, and points to the preferred utility of thiscombination reagent for this purpose.

Hybridization kinetics were measured in T³ relative to other reagents.The final salt concentrations of the reagents were as follows:

    ______________________________________                                        Reagent                                                                       Designation   Salt                                                            ______________________________________                                        E             2.5M GuSCN                                                      F             2.6M TMA.TFA                                                    G             4.2M TMA.SCN                                                    H             3.4M TMA, 2.1M SCN, 1.3M TFA                                    ______________________________________                                    

Results obtained in these experiments demonstrated that the besthybridization results were obtained in T³. Though hybridization inTMA.TFA was initially faster than in TMA.SCN, the level of hybridizationsubsequently fluctuated. The hybridization properties of T³ areadvantageously superior to those of TMA.SCN (in terms of rate and level)and to those of TMA.TFA (in terms of rate, level and stability); and allwere clearly and surprisingly superior to the hybridization propertiesof conventional GuSCN in terms of both rate of reaction and level ofsignal.

To test the specificity of hybridization in T³, two control experimentswere performed. In the first control, wherein the target was rRNA fromE. coli extracts, the number of counts captured was 7.5% of thereference experiment. In the second control, with no capture probe, thesignal level was 4.5% of the reference. These data reflect theadvantageous ability of the reagent to effect minimal non-specifichybridization and little non-specific (non-hybrid) binding.

As just described, hybridization proceeded faster and to a greaterextent in T³ than in TMA.TFA. These results were obtained with one typeof rRNA (from Salmonella extracts). To show that use of the novelreagents of the present invention was not limited to one rRNA species, aseries of experiments was carried out using three different species ofrRNA, three probe concentrations and the reagents of interest.

Reagent concentrations were: for TMA.TFA, 2.6M; for TMA.TFA/SCN, 3.4MTMA, 1.3M TFA, 2.1M SCN. From the resultant data the followingconclusions obtain:

1) In all the extracts examined, at a probe concentration of 100-500ng/ml, hybridization was faster in TMA.TFA/SCN than in TMA.TFA (onaverage, twice as fast in the first five minutes).

2) In all the extracts examined, at a probe concentration of 100-500ng/ml, hybridization proceeded to a greater extent in TMA.TFA/SCN thanin TMA.TFA (on average, 3 times as much after sixty minutes).

3) In TMA.TFA/SCN, a capture probe concentration of 100 ng/ml was justas good as a concentration of 500 ng/ml, in terms of both the rate andthe extent of hybridization.

As noted above, one very desirable, but not essential, property in ahybridization reagent is the ability to inhibit Ribonuclease. Data fromexperiments showed that TMA.TFA/SCN inhibits RNase A at concentrationsup to 0.125 μg/ml as well as, or better than, GuSCN. However, at higherRNase concentrations GuSCN was a better inhibitor of RNase A than wasTMA.TFA/SCN.

Next, Proteinase K was added to the reagent mixture to see if it wouldremain sufficiently active to decrease the RNase activity. The resultsdemonstrated that there was inhibition of the RNase activity by theProteinase K in TMA.TFA/SCN. For example, with RNase at 1.25 μg/ml, inTMA.TFA/SCN, Proteinase K reduced RNase activity by approximately 20%.

A more direct test of Proteinase K's utility (examining its ability todigest BSA) also demonstrated that the protease was active in T³. Thiswas in agreement with earlier described results where it was observedthat PK partially inhibited RNase A in T³. In another experiment it wasobserved that 25-50% of the Proteinase K activity was lost when theenzyme was stored at 37° C. in T³ over a period of two hours, whileunder similar circumstances in GuSCN, 80% of the enzymatic activity waslost.

A series of experiments was carried out to assess the ability of T³ tolyse bacterial cells. The data showed that T³ lysis efficiency could beadvantageously enhanced by the addition of SDS and Proteinase K, or to alesser extent SDS alone or SDS and DTT.

IV. TMA.SCN

Tetramethylammonium sulfate ( TMA!₂.SO₄) was prepared by neutralizingtetramethylammonium hydroxide with sulfuric acid. Tetramethylammoniumthiocyanate (TMA.SCN) was prepared by combining equimolar amounts ofTMA!₂.SO₄ and barium thiocyanate in solution, forming TMA.SCN (which issoluble) and barium sulfate (an insoluble precipitate):

     TMA!.sub.2.SO.sub.4 +Ba SCN!.sub.2 -->2 TMA.SCN!+BaSO.sub.4

Free barium ions in solution can be detected using rhodizonic acid,which forms an easily detectable insoluble red salt with the cation.(Feigl, F., Spot Tests, vol.I (1954, Elsevier, Amsterdam); Vogel, A. I.,Qualitative Inorganic Analysis (1979, Longman/Wiley, New York, N.Y.).This analytical test was used to determine the end-point of the reactionin the production of TMA.SCN: TMA.Sulfate was added to a solution of BaSCN!2, with periodic monitoring for Ba+, until the last of the bariumhad just precipitated from the solution. This was the equivalence pointof the reaction, with resulting equal molar concentrations of TMA andthiocyanate, and with barium sulfate precipitated out of solution. Theresulting mixture was centrifuged and the supernatant, which was aconcentrated solution (approximately 6M) of TMA.SCN, was decanted. Thissolution was buffered (b 100 mM Phosphate, 100 mM Borate, 50 mM EDTA;brought to pH 7.5 with TMA.OH) and has been found to be quite stable.

The concentration of thiocyanate can be determined by any one of avariety of standard analytical techniques. Colorimetric determination ofthe anion as a green copper/pyridine/thiocyanate complex (ibid.) wasdone. The concentration of TMA was the same as that of the thiocyanate(conservation of charge).

The optimal hybridization temperature (OHT) in TMA.SCN was shown to beabout 45°-50° C. It was further surprisingly discovered that thepre-binding of Riboprobe to target rRNA dramatically increased initialcapture probe-target binding rates, and moderately improved the finaldetection sensitivity. This means that a 1-2 hour pre-hybridization canbe advantageously employed to increase detection levels in this assay.(For example, a two-hour pre-hybridization, relative to ano-pre-hybridization assay, doubled the number of ribosomes detectedafter a 30-minute hybridization.) It should be noted that the number ofRiboprobes captured per cell in this series of experiments was extremelyhigh, up to nearly 28,000. Since the usual number of ribosomal targetsper cell is 10,000-20,000, this suggested that more than one Riboprobewas binding to each rRNA target upon long pre-incubation resulting inthe advantageous hyperhybridization phenomenon.

A series of experiments carried out in a variety of TMA.SCN saltconcentrations, from 2.5M to 4.2M, demonstrated that there was littlevariation of hybridization kinetics with TMA.SCN concentration, thoughin general it would seem that 3.5-4.2M salt is marginally preferred.Once again, the detection levels (up to 35,000 riboprobes captured percell) were excellent. Similar experiments done with 1.4M and 2.1MTMA.SCN indicated a less preferred sensitivity with those reagents.

Additional experiments showed that the capture probe concentration couldbe advantageously lowered to 0.5 μg/ml with little or no loss insensitivity, and that one could lower it to 0.2 μg/ml and achieve thesame level of detection after 30 minutes as in a 15-minute hybridizationwith 1.0 μg/ml probe, while in conventional GuSCN the concentrationcould not be less than 0.5-1.0 μg/ml without significantly reducingsignal strength.

The melting temperature (T_(d)) of a DNA-RNA hybrid in TMA.SCN was shownto be about 60° C. which was consistent with the optimal hybridizationtemperature (OHT) of about 50° C., since the OHT is usually slightlybelow the T_(d).

Resultant melting curves from experiments using 2.5M, 3.0M, 3.5 M, 4.0M,4.5M and 5.0M concentrations of TMA.SCN were remarkably similar,demonstrating advantageously that the melting behavior of the hybridswas independent of salt concentration over the range tested.

While additional experiments demonstrated inhibition of RNase activityby Proteinase K (at 2.5 mg/ml) in TMA.SCN, without Proteinase K, GuSCNdemonstrated superior RNase A inhibition.

Another series of experiments demonstrated that optimal lysis in TMA.SCNwhich was best achieved by use of an additive or additives such as SDS,DTT and/or Proteinase K. Alternatively, if one wished to avoid suchadditives and if TMA.SCN is to be used as the hybridization medium, thetarget cells can be first lysed with GuSCN, SDS/Proteinase K, or someother agent(s).

V. TMA.TFA

TMA.TFA was prepared by combining equimolar amounts of concentratedTrifluoroacetic acid (Sigma Chemical Co. or Pierce Chemical Co.) andTMA.OH (Sigma Chemical Co. or Southwestern Analytical Chemicals, Inc.)and buffering it with 0.1M phosphate, 0.1M borate, 0.05 mM EDTA(phosphoric acid, boric acid, and EDTA (free acid), neutralized withTMA.OH). Buffered TMA.TFA remained at a constant pH for at least 1 yearand its refractive index indicated a loss of only 2-3% of the solutesover the same period.

A hybridization reagent will advantageously strengthen A:T bondsrelative to G:C bonds such that all bond strengths are nearly equal. Inconventional 2.5M GuSCN, the A:T bond is so weak relative to the G:Cbond that the solution must be disadvantageously diluted three-fold toallow efficient capture of dA-tailed oligomers on oligo-dT-coatedmagnetic beads. One way of determining whether the A:T bond strength isincreased in a new reagent (relative to GuSCN) is to examine the captureof a dA-tailed probe (hybridized to a target and reporter probe) onoligo-t magnetic beads. Such experiments were carried out and it wassurprisingly discovered that capture can occur in TMA.TFA without anydilution of the TFA chaotrope.

An experiment was carried out to determine the stability of hybrids inTMA.TFA. The results demonstrated not only good hybrid stability ofTMA.TFA relative to GuSCN but in fact, surprisingly, that the number ofriboprobes detected in TMA.TFA hybridization was considerably higherthan that in GuSCN.

Other experiments showed that hybridization of a standard capture probeto its proper target was advantageously complete by the first time-point(five minutes) and independent of probe concentration (1.0 or 0.2 μg/ml)in TMA.TFA, while in GuSCN such hybridization was relatively slow anddependent on probe concentration, and did not reach anywhere near thesame level of completion by the end of 30 minutes. Hybridization of thesame probe to control RNA from heterologous extracts was also initiallymuch higher in TMA.TFA than in GuSCN. By doing a 15-minute hybridizationit was surprisingly found to be quite acceptable to use capture probe atconcentrations as low as 0.008-0.02 μg/ml. This was 50-125 times lesscapture probe than is ordinarily required with GuSCN and is indicativeof the unexpected, much faster hybridization kinetics in TMA.TFA than inGuSCN.

Still other results indicated that approximately 45° C. was the optimalhybridization temperature (OHT) in TMA.TFA. However, since OHT is afunction of probe (and hence hybrid) length, varying the probe lengthwould change the OHT. For example, if one wanted the OHT to be lower,one could conceivably shorten the probe. This is an important element offlexibility advantageously associated with this assay and the reagentsof the present invention.

Like TMA.SCN, TMA.TFA promotes rapid, efficient hybridization, and whileit is relatively ineffective in inactivating RNase, a reagent with thesegeneral properties could be advantageously employed in reactions such asnucleic acid amplification.

Inhibition of the RNase activity can be advantageously augmented by theaddition of Proteinase K. For example, RNase (0.125 μg/ml) was 92%,inactivated in the presence of the protease (2.5 mg/ml), compared tobeing 73% inactivated without the protease.

Other data indicated that ideal conditions for lysis in TMA.TFA areincubation at 65° C. with SDS, DTT and Proteinase K. However, evenbetter results may be obtained by preferably first lysing the targetcells with GuSCN or SDS/Proteinase K and then subsequently hybridizingin TMA.TFA.

TMA.TFA may also be advantageously employed to dramatically reduceaggregation by magnetic beads in the presence of stool samples and otherclinical samples. Ideally, the beads are resuspended in TMA.TFA (4M,buffered with phosphate, borate and EDTA; with 2% BSA and 10 μg/mlsingle-stranded sonicated calf thymus DNA) rather than the standard beadbuffer (0.1M Tris (pH 7.5), 0.5M NaCl, 10 mM EDTA, 0.5% (w/v) BSA(Fraction V), 0.5% Sodium lauryl sarcosine, 0.1% sodium azide plus 4%Saponin). With beads resuspended in TMA.TFA, bead aggregation wasdramatically reduced. These results are surprising, unexpected and ahighly advantageous property of the reagent particularly with thepreferred assays which use magnetic particles in combination withcapture probes to reduce background.

VI. TMA.TCA

TMA.TCA is ideally prepared by neutralizing highly concentrated

TCA (≈9.3M, or ≈1.5 g/ml) with TMA.OH. (This is an exothermic reactionwhich was found to proceed to completion only on ice with stirring.)Because TCA is volatile and reactive, its concentration drops with timeand the pH of the salt solution rises. This is ideally avoided since theefficiency of hybridization falls as the pH becomes less neutral. Tostabilize the solution's pH, it is preferably buffered with phosphate(0.1M) and borate (0.1M) advantageously resulting in pH stability over aperiod of at least one month. From the mass, volume and concentration ofstarting materials, and the volume of the final solution, the nominalconcentration of the final product was calculated to be about 3M.

Solid TMA.TCA may also be prepared by recrystallization with a largevolume of acetonitrile. This solid material was found to be stable, andmay be advantageously, subsequently dissolved in water to re-make abuffered solution of the salt.

The melting curve data of a dA-tailed 35-mer probe and its target RNA inTMA.TCA showed that the T_(d) was about 53°-55° C., with a breadth(.sup.Δ T_(3/4-1/4)) of about 2° C. The observed T_(d) generallycorrelated with three different chaotropes previously shown to lowerT_(d) by about 20° C. (Hamaguchi and Geiduschek (1962), Jour. Amer.Chem. Soc. 84, 1329-1338).

Hybridization of dA-tailed 35-mer Salmonella probe in 2.5M GuSCN wascompared with that in 3.0M TMA.TCA and the results indicated thathybridization was considerably faster and stronger in TMA.TCA than inGuSCN. It was also clear that no dilution of the chaotrope was requiredto effect capture on beads with the TMA.TCA. Experiments testing theconcentration of beads required to get 90% of the maximal captureefficiency in a specified amount of time showed that hybrids were stablefor two hours or longer in TMA.TCA and that capture occurred in highconcentrations (2.5M) of TMA.TCA at high temperatures (45° C.). Captureideally occurs in 3M TMA.TCA at about 37° C. Thus, capture canadvantageously occur without significant dilution of the chaotrope.

Additional experiments demonstrated that Proteinase K was advantageouslyactive in 3M TMA.TCA. 3M TMA.TCA also demonstrated preferredsolubilization of stool samples without aggregation of the magneticbeads. This was contrary to the effects seen with 0.83M GuSCN where aconsiderable amount of rapid aggregation was observed.

While preferred reagents and methods for their use have been describedherein, the present invention is not so limited and those skilled in theart will realize that many minor variations and modifications of thepresent invention are possible without departing from either the spiritor scope of the teaching and which are to be deemed equivalent andwithin the purview of the claims.

What is claimed is:
 1. A method for determining the presence of a targetnucleic acid in a specimen comprising the steps of;a) combining saidspecimen with an agent for disrupting molecular structure whereby saidtarget nucleic acid is exposed: b) contacting the result of step a) witha first capture nucleic acid probe specific for said target nucleic acidunder conditions allowing hybridization bonds therebetween to form ahybrid; c) capturing said hybrids on first solid support means byforming a bond between said first capture nucleic acid probe and saidfirst solid support means; d) washing said captured hybrids; e)contacting said captured hybrids with a first release buffer forreleasing said hybrids from said first solid support means andphysically separating said hybrids from said first solid support means;f) further contacting said hybrids with a second nucleic acid captureprobe specific for said target nucleic acid and different than saidfirst nucleic acid capture probe under conditions allowing bonds to formtherebetween; g) adding to said target nucleic acid a reagent to form amixture for effecting superstoichiometric labelling, said reagentcomprising a tetraalkylammonium cation and a chaotropic anion, of saidtarget nucleic acid by said labelled nucleic acid probe and incubatingsaid mixture under conditions permitting binding to occur wherebylabelled probe-target complexes are formed; h) capturing said labelledprobe-target complexes on second solid support means by forming a bondbetween said second capture nucleic acid probe and said second solidsupport means; i) contacting said captured labelled probe-targetcomplexes with a second release buffer for releasing said labelledprobe-target complexes from said second solid support means andphysically separating said labelled probe-target complexes from saidsecond solid support means; j) adding a labelled nucleic acid probecapable of binding to said target nucleic acid; k) optionally repeatingsteps h) and i); and l) detecting said labelled probe-target complexesand correlating the results of said detection with the presence oftarget nucleic acid in said sample.
 2. The method of claim 1 whereinsteps a) and b) are combined into a single step or steps e), f) and g)are combined into a single step or steps e), f), g) and h) are combinedinto a single step, or any combination of the foregoing are combined. 3.The method of claim 1 wherein:a) in said first release buffer the bondsbetween said first nucleic acid capture probe and said first solidsupport means are stronger than the bonds between said first nucleicacid capture probe said nucleic acid target whereby said target nucleicacid can be released from said first solid support means while saidfirst nucleic acid capture probe remains bound to said first solidsupport means, and b) in said second release buffer the bonds betweensaid second nucleic acid capture probe and said second solid supportmeans are weaker than the bonds between said second nucleic acid captureprobe and said nucleic acid target whereby said target nucleic acidremains bound to said second nucleic acid capture probe when said secondnucleic acid capture probe is released from said second solid phasemeans.
 4. The method of claim 3 wherein the bonds formed between saidfirst nucleic acid capture probe and said first solid support means, andbetween said second nucleic acid capture probe and said second solidsupport means comprise:a) a first member comprising a RNA or DNAoligomeric repeating sequence, said repeating sequence being no longerthan approximately four nucleotides, and b) a second member comprising arepeating sequence complementary to said first member whereby said firstor second nucleic acid capture probe comprises said first or secondmember, and said first or second solid support means comprises the otherof said first or second member.
 5. The method of claim 4 wherein saidfirst release buffer increases the strength of poly(dA)-poly(dT) bonds,poly(dA)-oligo(dT) bonds, or poly(dT)-oligo(dA) bonds, relative to bondsbetween mixed base sequences; or said second release buffer decreasesthe strength of poly(dA)-poly(dT) bonds, poly(dA)-oligo(dT) bonds, orpoly(dT)-oligo(dA) bonds relative to bonds between mixed-base sequencesor both.
 6. The method of claim 5 wherein said first release buffercomprises a tetraalklyammonium salt and said second release buffercomprises a chaotrope.
 7. The method of claim 1 wherein said secondnucleic acid capture probe and said second solid phase means form bondsdifferent in composition than the bonds formed between said firstnucleic acid capture probe and said first solid phase means.
 8. Themethod of claim 7 wherein the bonds between said first nucleic acidcapture probe and said first solid phase means are the result ofcomplementary oligomer repeating sequences, wherein each repeatedsequence is no longer than approximately four nucleotides; and whereinthe bonds between said second nucleic acid capture probe and said secondsolid phase means are the result of bonds between a first member of aspecific binding pair and a second member of said specific binding pair,an antigen and an antibody, biotin and avidin, or biotin andstreptavidin.
 9. The method of claim 7 wherein the bonds between saidfirst nucleic acid capture probe and said first solid phase means arethe result of complementary oligomer repeating sequences, wherein eachrepeated sequence is no longer than approximately four nucleotides. 10.The method of claim 1 wherein said labeled nucleic acid probe directlyor indirectly comprises a label selected from an RNA moleculeamplifiable by replication using a Q-beta replicase enzyme, isotope,chemiluminescent compound, bioluminescent compound, an enzyme having adetectable activity, a hapten, a magnetic particle and an opticallydetectable particle.
 11. A method for determining the presence orabsence of a target nucleic acid in a mixture of nucleic acids in asample, comprising the steps of:a) disrupting molecular structure ofsaid sample by sonicating, bead-beating, or contacting said samplewithin a chemical agent, thereby exposing said target nucleic acid ifpresent; b) adding to the result of step a) a nucleic acid capture probespecific for said target nucleic acid under conditions allowinghybridization therebetween to form a hybrid; c) immobilizing saidhybrids on solid support means; d) separating immobilized hybrids formedin step c); e) releasing said immobilized hybrids from said solidsupport means; f) contacting said released hybrids with a labelednucleic acid probe capable of binding to said target nucleic acidwherein said contacting step is carried out in the presence of a reagentwhich promotes superstoichiometric labelling of said nucleic acid saidreagent comprising a tetraalkylammonium cation and a chaotropic anion;and g) detecting labelled nucleic acid if present.